Conjugates of therapeutic or cytotoxic agents and biologically active peptides

ABSTRACT

The invention features conjugates of therapeutic or cytotoxic agents and biologically active peptides and methods of use thereof.

FIELD OF THE INVENTION

The invention relates to conjugates of therapeutic or cytotoxic agentsand biologically active peptides and uses therof.

BACKGROUND OF THE INVENTION

The use of carbamate compounds as prodrugs is well known. Carbamatecompounds are well suited for prodrug design because they can be used toregenerate the parent drug whether the point of connection to a vectormolecule is a hydroxyl group or an amine. Carbamate prodrugs based onintramolecular cyclizations have been reported since the late 1980s. Forexample, U.S. Pat. No. 4,812,590 (which corresponds to EP 0 296 811)discloses derivatives of 4-hydroxyanisole carbamate as prodrugs for thedelivery and concentration of 4-hydroxyanisole in melanomas. Vigroux etal. (J. Med. Chem. 38, 3983-3994, 1995) disclose prodrugs ofacetaminophen which incorporate N-(substituted 2-hydroxyphenyl)- andN-(substituted 2-hydroxypropyl)carbamates.

An optimal prodrug design is one in which the drug is conjugated to abiologically active vector such that the conjugate is completely stablein circulation, but releases the therapeutic agent cleanly wheninternalized into target cells. Various types of linker technologiesattempt to achieve this goal by different chemical means. Two types ofbiodegradation phenomena, passive hydrolysis and enzymatic hydrolysis,are typically considered. Passive hydrolysis occurs when the moleculedegrades by simple chemical decomposition; esters, carbonates, amidesand urethanes are all susceptible to varying degrees of hydrolysis withthe following order of stability at basic pH:urethanes >amides>carbonates>esters. Enzymatic hydrolysis occurs inplasma circulation as well as upon internalization. If the desiredresult is to transport the conjugate to the target cell, it is desirablethat the conjugate be very resistant to plasma enzymes that coulddegrade the complex en route. Commonly used ester derivatives are aparticularly poor choice of linker because peripherally circulatingcompound is rapidly attacked by ubiquitous esterases.

Another linker strategy capitalizes on pH differences existing betweenthe inside and outside of cells. Lysosomes, which are responsible fordegrading molecules within cells, have a pH of 4-6, while plasma andextracellular fluid has a pH of 7.4. A linker that is designed todegrade at pH 5, but is stable at pH 7, may be useful for delivering theconjugate to cells, assuming that the conjugate does not undergo passiveor enzymatic hydrolysis in plasma. However, this linker strategy is notoptimal for peptide conjugates because most of the work-up andpurification of synthetic peptides is done in acidic media.

Still other chemical linkers attempt to capitalize on specific enzymesfound in lysosomes. For example, many peptide sequences are stable inplasma but are specifically cleaved by lysosomal enzymes.

The toxic side effects of many of these therapies, as well as standardtreatments of neoplastic disease, effectively limit the amount of activeagent that may be administered to a patient. Additionally, many activeagents cause organ-specific toxicities, further limiting the dose thatmay be delivered to the target tissue. For instance, the cardiotoxicityof many anthracycline family members reduces the maximum therapeuticdose available for this group of chemotherapeutic agents. Targeted drugdelivery of various therapeutic agents can lower toxicity in normaltissue and increase the efficacy of treatment by allowing concentratedlocalized effects on specific tissues.

Somatostatin, bombesin, and other biologically active peptide analogshave been used to detect tumor cells that overexpress receptors specificfor these peptides (see, e.g., Denzler and Reubi, Cancer 85(l):188-198,1999). Moreover, somatostatin, bombesin and many other biologicallyactive peptide agonist analogs are rapidly internalized after binding totheir receptors (see, e.g., Lukinius et al, Acta Onc. 38:383-387, 1999).This internalization of the peptide analogs may result in translocationto the cell nucleus (Chen et al., Am. J. Physiol. Renal Physiol., 279:F440-F448, 2000).

Somatostatin analogs bind specific somatostatin receptor subtypes thatare present on the surface of specific normal or diseased tissues.Somatostatin receptors are up-regulated in specific diseased tissues,including inflammatory bowel diseases, rheumatoid arthritis, and avariety of tumor types, as well as blood vessels supplying many tumors(Denzler and Reubi, Cancer, 85:4188-198, 1999). Similarly, receptorsspecific for another biologically active peptide, Substance P, can beup-regulated in various diseases.

At least five somatostatin receptors subtypes have been characterized,and tumors can express various receptor subtypes. (Shaer, et al., Int.3. Cancer 70:530-537, 1997). Naturally occurring somatostatin and itsanalogs exhibit differential binding to these receptor subtypes,allowing precise targeting of a peptide analog to specific diseasedtissues.

The physical and chemical properties of many cytotoxic agents make drugconjugation to biologically active peptides, such as somatostatin andbombesin, problematic. For example, the drug may reduce the specificityof binding or the biological activity of the peptide analog, limitingits effectiveness as a targeting agent. Additionally, therapeutic andcytotoxic agents may have chemical properties that promote accumulationof drug-peptide analogs in certain organs, increasing toxicity andreducing efficacy. Effective means to link cytotoxic agents to atargeting agent such as a biologically active peptide or an antibody,while retaining the activity of each component are needed to maximizetherapeutic effects, while minimizing toxicity.

SUMMARY OF THE INVENTION

The invention features conjugates of a therapeutic agent or a cytotoxicagent and a targeting moiety. In particular, the invention featuresconjugates having a cleavable chemical linker which reduces the releaseof therapeutic or cytotoxic agent in circulation, thereby rendering theactive agent component of the conjugate more readily internalized by acell, and more readily subject to active control of release rate insidethe cell.

Accordingly, in one aspect, the present invention features biologicallyactive peptides conjugated to chemical compounds through a carbamatelinkage. Conjugates of the present invention provide numerousadvantages, such as retention of the biological activity of the peptide,enhanced stability of the conjugate in plasma, and intracellular releaseof the attached compound. Preferred peptides for use with conjugates ofthe invention include somatostatin, somatostatin analogs, bombesin,bombesin analogs, KiSS peptides and analogs, urotensin II peptides andanalogs, GnRH I and II peptides and analogs, octreotide, depreotide,vapreotide, vasoactive intestinal peptide (VIP), cholecystokinin (CCK),insulin-like growth factor (IGF), RGD-containing peptides,melanocyte-stimulating hormone (MSH) peptide, neurotensin, calcitonin,peptides from complementarity determining regions of an antitumorantibody, glutathione, YIGSR (leukocyte-avid peptides, e.g., P483H,which contain the heparin-binding region of platelet factor-4 (PF-4) anda lysine-rich sequence), atrial natriuretic peptide (ANP), β-amyloidpeptides, delta-opioid antagonists (e.g., [¹²⁵I]ITIPP(psi)[H-Tyr(3′I)-Ticpsi[CH2NH]Phe-Phe-OH]; ITIPP(psi)), annexin-V,endothelin, interleuking (IL)-1, IL-1ra, IL-2, and IL-8, leukotriene B4(LTB4), chemotactic peptides (e.g.,N-formyl-methionyl-leucyl-phenylalanine-lysine (fMLFK)), GP IIb/IIIareceptor antagonists (e.g., DMP444), epidermal growth factor, humanneutrophil elastase inhibitor (e.g., EPI-HNE-2 and EPI-HNE-4), plasmininhibitor, antimocrobial peptides, apticide (e.g., P280 and P274),thrombospondin receptor (including analogs such as TP-1300), bitistatin,pituitary adenylyl cyclase type I receptor (PAC1), fibrin α-chain, andderivatives and analogs thereof. Also included are peptides derived froma phage display library, and conservative substitutions thereof, thatare targeted to a cell or tissue in the body of a mammal (e.g., diseasedtissue, such as a tumor or a proliferative angiogenic blood vessel; see,e.g., Aina et al., Biopolymers 66:184-199, 2002). Such peptides areuseful for specifically targeting therapeutic agents and cytotoxicagents to a cell, such as a cancer cell, or to a tissue in the body of amammal (e.g., the delivery of anti-apoptotic drugs to cardiac or braintissue), or to selectively target white blood cells or tuberclesinfected with tuberculosis. For example, when somatostatin or bombesinis used as the biologically active peptide in a conjugate of theinvention, the therapeutic or cytotoxic agent may be targeted to acancer cell that expresses a somatostatin receptor or a bombesinreceptor. The invention also features antibodies (e.g., a monoclonalantibody) or a fragment thereof, that can be linked to the conjugate ofthe invention. As discussed above with respect to peptides, when anantibody is incorporated in a conjugate of the invention, thetherapeutic or cytotoxic agent may be targeted to specific antibodybinding sites.

Conjugates of the first aspect of the invention have the followinggeneral formula:

wherein

X is a cytotoxic agent or therapeutic agent;

n is an integer from 0 to 6, wherein (CH₂)_(n) is substituted orunsubstituted, a straight or branched chain, or is an alkyl, alkenyl,alkynyl, cyclic, heterocyclic, aromatic, or heteroaromatic group;

R is N(R₁R₂), OR₁, or SR₁ where R₁ and R₂ are, independently, hydrogenor a lower alkyl group;

Y is an hydrophilic spacer sequence, or is omitted;

Z is a linking peptide, or is omitted; and

Q, when present, is a targeting moiety.

In this and the other aspects of the invention, the alkyl grouppreferably contains 1-8 carbon atoms; the alkenyl group preferablycontains 1-X carbon atoms; the alkynyl group contains 1-X carbon atoms;the cyclic group contains . . .

A second aspect of the invention features a conjugate having theformula:

wherein

X is a cytotoxic agent or therapeutic agent;

n is an integer from 0 to 6, wherein (CH₂)_(n) is substituted orunsubstituted, a straight or branched chain, or is an alkyl, alkenyl,alkynyl, cyclic, heterocyclic, aromatic, or heteroaromatic group;

R is N(R₁R₂), OR₁, or SR₁ where R₁ and R₂ are, independently, hydrogenor a lower alkyl group, and wherein R₃ is a NH(CH₂)mSH group (where m=2to 6), a D or L cysteine, a benzophenone (e.g.,p-benzoyl-phenylalanine), or an OH group. A conjugate compound havingthis formula can be used to link to a peptide, protein, or antibodythrough the thiol, carboxylate, or photoactive benzophenone containingR₃ group. In addition, a peptide or protein can be easily prepared bysynthesizing the peptide or protein directly onto the compound. Forexample, the compound can be provided such that the R group is a tBocprotected primary or secondary amino group, and can be added to thepeptidyl resin as described in the examples provided below. Peptides,proteins, and antibodies can also be attached to the compound throughthe use of known thiol-reactive chemical reactions when the R₃ groupcontains a thiol group, or by a photoreactive reaction when R₃ containsa benzophenone moiety (see, e.g., Greg T. Hermanson, “BioconjugateTechniques” p. 146-152, 1996).

In preferred embodiments of all aspects of the invention, X is acytotoxic agent selected from camptothecin, homocamptothecin,colchicine, combretastatin, dolistatin, doxorubicin, methotrexate,podophyllotoxin, rhizoxin, rhizoxin D, a taxol, paclitaxol, CC1065, or amaytansinoid and derivatives and analogs thereof. For example, thecytotoxic agent camptothecin is linked through its single free hydroxylgroup to a carbamate linker. In these embodiments of the invention, n ispreferably 2 and R is preferably NH₂.

Y is an hydrophilic spacer sequence that may be, preferably, a peptidethat increases the biodistribution of the conjugate, or a hydrophilicpolymer. For example, Y may be a peptide sequence that increases thehydrophilic biodistribution of the biologically active peptideconjugate. In a preferred embodiment, Y has the formula U(V-V)_(n),wherein U is D-Pro, L-Pro, D-4-OH-Pro, L-4-OH-Pro, Sarcosine, Lys, Orn,Dab, Dap, 4-NH₂-Phe, or (NH₂—(CH₂)_(m)—COOH), where m=2-10, inclusive,or is deleted; each V is independently selected from the groupconsisting of: D-Ser, L-Ser, D-Thr, L-Thr, D-Gln, L-Gln, D-Asn, L-Asn,D-4-OH-Pro, or L-4-hydroxy-Pro; and n=1-50, inclusive. In anotherpreferred embodiment, each V is independently D-Ser or L-Ser. In anotherpreferred embodiment, at least one V is a D-amino acid.

Y may be a hydrophilic polymer. For example, Y may be polyethyleneglycol, polyvinyl acetate, polyvinyl alcohol, HPMA(N-(2-hydroxypropyl)methacrylamide) or HPMA copolymers, α,β-poly(N-hydroxyethyl)-DL-aspartamide (PHEA), or α,β-poly(N-hydroxypropyl)-DL-aspartamide.

Z is a linking peptide that preserves at least fifty percent of thebiological activity of Q, when Z is bonded to Q through the terminal orside-chain amino group of Q. Generally, Z may be a peptide of 2, 3, 4,or 5 residues. Z has the formula: A-B-C-E-F, where A is D-Lys, D-Tyr,D-Ser, or L-Ser, or deleted; B is D-Lys or D-Tyr, or is deleted; C isLys, Ser, hSer, Thr, Nle, Abu, Nva, (2,3, or 4) 3-pyridyl-Ala (Pal),Orn, Dab, Dap, 4-NH₂-Phe, D-4-OH-Pro, or L-4-OH-Pro, or is deleted; E isD-Lys, D-Tyr, D-Ser, D-4-OH-Pro, L-4-OH-Pro, 3-iodo-D-Tyr, 3-5diido-D-Tyr, 3-astatine-D-Tyr, 3-5 astatine-D-Tyr, 3-bromo-D-Tyr, 3-5dibromo-D-Tyr, D-Asn, L-Asn, D-Asp, L-Asp, D-Glu, L-Glu, D-Gln, orL-Gln; and F is D-Lys, D-Tyr, D-Ser, L-Ser, D-4-OH-Pro, L-4-OH-Pro,3-iodo-D-Tyr, 3-5 diido-D-Tyr, 3-astatine-D-Tyr, 3-5 astatine-D-Tyr,3-bromo-D-Tyr, 3-5 dibromo-D-Tyr, D-Asn, L-Asn, D-Asp, L-Asp, D-Glu,L-Glu, D-Gln, or L-Gln; provided that when A, B, C, and E are Tyr, Tyr,Lys, and Tyr, respectively, F is not Lys; and when A, B, C, and E, areLys, Tyr, Lys, and Tyr, respectively, F is not Tyr or Lys; and when Aand B are deleted, and C and E are Lys and Tyr, respectively, F is notTyr or Lys. In some peptides, it may be preferable that Z is absent.

In other embodiments, Z has the formula (when A, B, and C are deleted):

E-F

where E is D-Lys, D-Tyr, D-Ser, D-4-OH-Pro, L-4-OH-Pro, 3-iodo-D-Tyr,3-5 diiodo-D-Tyr, 3-astatine-D-Tyr, 3-5 astatine-D-Tyr, 3-bromo-D-Tyr,3-5 dibromo-D-Tyr, D-Asn, L-Asn, D-Asp, L-Asp, D-Glu, L-Glu, D-Gln, orL-Gln; and F is D-Lys, D-Tyr, D-Ser, L-Ser, D-4-OH-Pro, L-4-OH-Pro,3-iodo-D-Tyr, 3-5 diiodo-D-Tyr, 3-astatine-D-Tyr, 3-5 astatine-D-Tyr,3-bromo-D-Tyr, 3-5 dibromo-D-Tyr, D-Asn, L-Asn, D-Asp, L-Asp, D-Glu,L-Glu, D-Gln, or L-Gln.

In preferred embodiments, Z is D-Ser-Nle-D-Ser-D-Ser (SEQ ID NO: 1),D-Ser-Lys-D-Ser-D-Ser (SEQ ID NO: 2), D-Ser-Lys-D-Tyr-D-Tyr (SEQ ID NO:3), D-Ser-Lys-D-Tyr-D-Ser (SEQ ID NO: 4), D-Ser-Ser-D-Lys-D-Ser (SEQ IDNO: 5), D-Ser-Ser-D-Lys-Ser (SEQ ID NO: 5), D-Ser-Nle-D-Tyr-D-Ser (SEQID NO: 6), D-Ser-Pal-D-Tyr-D-Ser (SEQ ID NO: 7), D-Ser-Thr-D-Tyr-D-Ser(SEQ ID NO: 8), Lys-D-Ser-D-Ser (SEQ ID NO: 9), Ser-D-Lys-D-Ser (SEQ IDNO: 10), Ser-D-Lys-Ser (SEQ ID NO: 10), Nle-D-Tyr-D-Ser (SEQ ID NO: 11),Lys-D-Tyr-D-Ser (SEQ ID NO: 12), Pal-D-Lys-D-Ser (SEQ ID NO: 13),Thr-D-Tyr-D-Ser (SEQ ID NO: 14), D-Ser-D-Lys, D-Ser-D-Tyr, D-Lys-D-Lys,D-Lys-D-Tyr, or D-Tyr-D-Lys.

Q is a targeting moiety, such as a biologically active peptide or phagederived peptides. In preferred embodiments, Q is a peptide, such assomatostatin, a somatostatin analog, bombesin, a bombesin analog, or anantibody, such as a monoclonal antibody. Preferred biologically activepeptide or targeting moieties are internalized by select cells via anactive internalization process, such as when binding to a G-proteincoupled receptor or somatostatin type 2 receptors (SSTR2).

Another aspect of the invention features a method for the treatment of adisease or modification of a biological function. The method includesadministering to a warm-blooded animal in need thereof, e.g., a human, atherapeutically effective or biological function modifying amount of aconjugate of the invention. One of skill in the art will recognize thatthe particular conjugate used will depend on the disease state to betreated or the biological system to be modified. In particular, oneskilled in the art will be able to select a particular targeting moietyand cytotoxic or therapeutic agent to prepare a conjugate of theinvention which has specificity for the treatment of the disease or isable to modify the biological function desired. In preferredembodiments, the disease is a tumor of the lung, breast, brain, eye,prostate or colon or a tumor of neuroendocrine origin (e.g., carcinoidsyndrome). The disease is also a condition that results from or causesproliferation of angiogenic blood vessels.

By “administration” or “administering” is meant a method of giving adosage of a pharmaceutical composition to a mammal, where the method is,e.g., topical, oral, intravenous, intraperitoneal, or intramuscular. Thepreferred method of administration can vary depending on variousfactors, e.g., the components of the pharmaceutical composition, site ofthe potential or actual disease and severity of disease.

In the generic descriptions of compounds of this invention, the numberof atoms of a particular type in a substituent group is generally givenas a range. For example, an alkyl group containing from 1 to 8 carbonatoms or C₁₋₈ alkyl. Reference to such a range is intended to includespecific references to groups having each of the integer number of atomswithin the specified range. For example, an alkyl group from 1 to 8carbon atoms includes each of C₁, C₂, C₃, C₄, C₅, C₆, C₇, and C₈. A C₁₋₈heteroalkyl, for example, includes from 1 to 8 carbon atoms in additionto one or more heteroatoms. Other numbers of atoms and other types ofatoms may be indicated in a similar manner.

As used herein, “alkyl”, is intended to include aliphatic branched orstraight chain hydrocarbon group and combinations thereof. An alkyl isoptionally substituted with one or more substituents which may be thesame or different. An alkyl group contains 1 to 20 carbon atoms,preferably 1 to 8 carbon atoms. Examples include methyl, ethyl,n-propyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isoamyl,hexyl, octyl and the like. By “lower alkyl” is meant a branched orstraight chain alkyl group having fewer than 11 carbon atoms, preferablya C₁-C₈ alkyl. By “lower alkylamide” is meant a lower alkyl group asdescribed above substituted with one or more amide-containing groups. Asused herein, the terms “alkyl” and the prefix “alk-” can also refer tocyclic groups, i.e., cycloalkyl and cycloalkenyl groups. Cyclic groupscan be monocyclic or polycyclic and preferably have from 3 to 8 ringcarbon atoms, inclusive.

The term “alkenyl”, alone or in combination, means a straight-chain orbranched-chain hydrocarbon having one or more double bonds andcontaining from 2 to 20 carbon atoms, preferably from 2 to about 8carbon atoms. The term “alkynl”, alone or in combination, means astraight-chain or branched-chain hydrocarbon having one or more triplebonds and containing from 2 to about 20 carbon atoms, preferably from 2to 8 carbon atoms.

By “heteroalkyl” is meant a branched or unbranched group in which one ormore methylenes (—CH₂—) are replaced by nitrogen, oxygen, sulfur,carbonyl, thiocarbonyl, phosphoryl, sulfonyl, or NR, where R is analkyl. Some examples include tertiary amines, ethers, thioethers,amides, thioamides, carbamates, thiocarbamates, phosphoramidates,sulfonamides, and disulfides. A heteroalkyl may optionally includemonocyclic, bicyclic, or tricyclic rings, in which each ring desirablyhas three to six members. The heteroalkyl group may be substituted orunsubstituted.

By “analog” is meant a molecule that differs from, but is structurally,functionally, and/or chemically related to the reference molecule. Theanalog may retain the essential properties, functions, or structures ofthe reference molecule. Most preferably, the analog retains at least onebiological function of the reference molecule. Generally, differencesare limited so that the structure or sequence of the reference moleculeand the analog are similar overall. A peptide analog and its referencepeptide may differ in amino acid sequence by one or more substitutions,additions, and/or deletions, in any combination. A substituted orinserted amino acid residue may or may not be one encoded by the geneticcode of the peptide analog. An analog of a peptide or polypeptide may benaturally occurring, such as an allelic variant, or it may be a variantthat is not known to occur naturally. Non-naturally occurring analogs ofpeptides may be made by direct synthesis, by modification, or bymutagenesis techniques.

By “biologically-active peptide” is meant any naturally occurring,modified, or synthetic peptide that is involved in a biological processor function. Examples of biologically active peptides include, but arenot limited to: hormones, growth factors, neurotransmitters, antigens,antibodies, or fragments thereof.

The term “bombesin peptide” is meant bombesin, or an analog thereof,having at least one biological activity of native bombesin; preferably,this activity is the ability to specifically bind to one or all of thethree known bombesin receptor subtypes on a bombesin receptor-bearingcell. Bombesin analogs include, but are not limited to, peptidesselected from the group containing the octapeptide G-Trp-H-I-His-J-K-NHV(SEQ ID NO: 15), wherein G is Gln, Asn, Nle, or Nva; H is Ava, Gly, Leu,Val, Ile, Nle, or Nva; I is β-Ala, 4-aminobutyric acid, Gly, Ala, D-Ala,N-Me-Ala, or N-Me-D-Ala; J is Phe, Tyr, 4-Cloro-Phe, 4-Fluoro-Phe,4-Bromo-Phe, 4-NO₂-Phe, Ala, Gly, Leu, Val, Ile, Nle, or Nva; K is Met,Phe, Tyr, 4-Cloro-Phe, 4-Fluoro-Phe, 4-Bromo-Phe, 4-NO₂-Phe, Ala, Gly,Leu, Val, Ile, Nle, or Nva; and N represents an amide or a N-alkylamideand V is H or a lower alkylamide.

By “cyclic” is meant a hydrocarbon group having from 3 to 10 carbons andmore preferably from 3 to 6 carbons.

By “cytotoxic agent” is meant any naturally-occurring, modified, orsynthetic compound that is toxic to tumor cells. Such agents are usefulin the treatment of neoplasms, as well as inflammatory diseases,autoimmune disorders, and in the treatment of other symptoms or diseasescharacterized by cell proliferation or a hyperactive cell population.Cytotoxic agents include, but are not limited to, alkylating agents,antibiotics, antimetabolites, tubulin inhibitors, topoisomerase I and IIinhibitors, hormonal agonists or antagonists, or immunomodulators.Cytotoxic agents may be cytotoxic when activated by light or infrared(Photofrin, IR dyes; Nat. Biotechnol. 19(4):327-331, 2001), may operatethrough other mechanistic pathways, or be supplementary potentiatingagents.

By “hydrophilic biodistribution” is meant the affinity of the peptideagents of the invention for the bodily fluids of a subject administeredthe peptide agents (e.g., blood, cerebrospinal fluid, urine, or otherbodily fluids), such that the peptide agents distribute throughout thebody of the subject, but are rapidly secreted in the urine via thekidney, while avoiding uptake by peripheral organs such as liver, gallbladder, and kidney proximal tubules.

By “hydrophilic polymer” is meant a naturally-occurring or syntheticwater-soluble polymer optionally modified that alters thebiodistribution of a peptide agent of the invention. Examples of suchpolymers include, but are not limited to poly(ethylene glycol) (PEG),poly(vinyl alcohol) (PVA), polyvinyl acetate, dextran, hydroxyelthylstarch, gelatin, PVP, PHPMA, α, β-poly[N(2-hydroxyethyl)-DL-aspartamide(PHEA), polysuccinamide (PSI), α,β-poly(N-hydroxypropyl)-DL-aspartamide, etc. These polymers may bemodified by, for example, succinylation (negative charge), partialhydrolysis of PSI (carboxylic groups), or reaction with compounds to addamino- or carboxyl-containing groups, etc. Such optional modificationsmay increase or change the hydrophilicity of the polymer, or enablecoupling to the peptide or cytotoxic or therapeutic agent of theinvention. Such polymers and modifications are known in the art and aredescribed in, for example, Yamoaka et al., J. Pharmacol. Sci.83:601-606, 1994; Rypacek et al., Pflugers Arch. 392:211-217, 1982;Yamoaka et al., J. Pharm. Pharmacol. 47:479-486, 1995; Francesco,Bioconjugate Chemistry 9(4):418-450, 1998; Duncan and Spreafico, Clin.Pharmacokinet. 27(4):290-306,1994; each of which is hereby incorporatedby reference in its entirety.

By “hydrophilic spacer sequence” is meant an hydrophilic peptide orhydrophilic polymer that increases biodistribution of a compound of theinvention, such as by inhibiting peripheral accumulation and/orpromoting renal clearance. Examples of hydrophilic spacer sequences foruse with the present invention are provided herein.

By “peptide” is meant any polypeptide, peptide (including cyclic orbranched peptides), or protein comprising two or more amino acids joinedto each other by peptide bonds or modified peptide bonds. As usedherein, peptide refers to short chains, commonly referred to aspeptides, oligopeptides or oligomers, and to longer chains, up to about100 residues in length. Peptides may contain amino acids other than the20 gene-encoded amino acids, and linkages other than peptide bonds.Peptides include amino acid sequences modified either by naturalprocesses, or by chemical modification techniques that are well known inthe art. Modifications may occur anywhere in a polypeptide, includingthe peptide backbone, the amino acid side-chains, and the amino orcarboxyl termini.

The notations used herein for the peptide amino acid residues are thoseabbreviations commonly used in the art. The less common abbreviationsAbu, Ava, β-Ala, hSer, Nle, Nva, Pal, Dab, and Dap stand for2-amino-butyric acid, amino valeric acid, beta-aminopropionic acid,homoserine, norleucine, norvaline, (2, 3, or 4) 3-pyridyl-Ala,1,4-diaminobutyric acid, and 1,3-diaminopropionic acid, respectively. Inall aspects of the invention, it is noted that when amino acids are notdesignated as either D- or L-amino acids, the amino acid is either anL-amino acid or could be either a D- or L-amino acid, unless the contextrequires a particular isomer.

By “somatostatin peptide” is meant a somatostatin, or an analog thereof,having at least one biological activity of native somatostatin;preferably, this activity is the ability to specifically bind to asomatostatin receptor on a somatostatin receptor-bearing cell. Many suchanalogs having biological activity are known and have been described,for example, in Hornik et al., U.S. Pat. No. 5,770,687; Coy et al., U.S.Pat. No. 5,708,135; Hoeger et al., U.S. Pat. No. 5,750,499; McBride etal., U.S. Pat. No. 5,620,675; Coy et al., U.S. Pat. No. 5,633,263; Coyet al., U.S. Pat. No. 5,597,894; Taylor et al., U.S. Pat. No. 5,073,541;Coy et al., U.S. Pat. No. 4,904,642; Dean, U.S. Pat. No. 6,017,509;Hoffman et al., WO 98/47524; and A. E. Bogden, U.S. Pat. No. 5,411,943,each of which is hereby incorporated by reference in its entirety.

By “targeting moiety” is meant any molecule that specifically binds orreactively associates or complexes with a receptor or other receptivemoiety associated with a given target cell population. This cellreactive molecule, to which the drug reagent is linked via the linker inthe conjugate, can be any molecule that binds to, complexes with orreacts with the cell population sought to be therapeutically orotherwise biologically modified and, which possesses a free reactiveamine group or can be modified to contain a such an amine group. Thecell reactive molecule acts to deliver the conjugate, and, thus, thecytotoxic or therapeutic agent to the particular target cell populationwith which the ligand reacts. Such molecules include, but are notlimited to, large molecular weight proteins (generally greater than10,000 daltons) such as, for example, antibodies, smaller molecularweight proteins (generally, less than 10,000 daltons), polypeptide orpeptide ligands, and non-peptidyl ligands.

By “therapeutic agent” is meant any compound that is used in thedetection, diagnosis or treatment of human disease. Such compounds maybe naturally-occurring, modified, or synthetic. Therapeutic agents maypromote or inhibit any biological process implicated in a human diseasepathway. Preferred disease targets include, but are not limited to,inflammatory bowel disease, rheumatoid arthritis, neoplastic cellsapoptosis, cardiac tissues, or aberrantly proliferating cells, carcinoidsyndrome, acromegaly, tuberculosis, and angiogenesis that causesinappropriate proliferation of blood vessels (e.g., macular degenerationwhich results from excess angiogenesis in the eye). A therapeutic agentmay be, for example, antineoplastic, including cytotoxic. Antineoplasticagents may be alkylating agents, antibiotics, antimetabolites, hormonalagonists or antagonists, tubulin inhibitors, topoisomerase I and IIinhibitors, anti- or pro-apoptotic agents, or immunomodulators.Antineoplastic agents may operate through other mechanistic pathways, orantineoplastic agents may be supplementary potentiating agents.

By “treating” is meant administering a pharmaceutical composition forprophylactic and/or therapeutic purposes. To “prevent disease” refers toprophylactic treatment of a patient who is not yet ill, but who issusceptible to, or otherwise at risk of, a particular disease. To “treatdisease” or use for “therapeutic treatment” refers to administeringtreatment to a patient already suffering from a disease to amelioratethe disease and improve the patient's condition. Thus, in the claims andembodiments, treating is the administration to a mammal either fortherapeutic or prophylactic purposes.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram demonstrating the on-resin synthesis of a BINARgroup-containing somatostatin analogue and attachment of camptothecinvia a carbamate linking group followed by removal from the resin to givecompound 2. Basic conditions or enzymatic attack results innucleophile-assisted release of free camptothecin.

FIG. 2 is a graph demonstrating the dose-response curve for conjugates1-6 and showing the ability of conjugates 1-6 to kill somatostatinreceptor subtype 2 (SSTR2) positive IMR-32 cells after incubation for 3days using a cell viability MTT assay.

DETAILED DESCRIPTION OF THE INVENTION

The present invention features conjugates of cytotoxic or therapeuticagents and biologically peptides. These conjugates provide manyadvantages for the administration of such cytotoxic or therapeuticagents. Conjugates of the invention may be directed to target sites orspecific cells, resulting in effective internalization of the cytotoxicor therapeutic agent In addition, conjugates of the invention exhibitreduced release of cytotoxic or therapeutic agent in circulation.Conjugates of the invention may be modified as desired to adjust therelease of cytotoxic or therapeutic agent Conjugates of the inventionhave the following general formula:

wherein X is a cytotoxic agent or therapeutic agent;

n is an integer from 0 to 6, wherein (CH₂)_(n) is substituted orunsubstiuted, straight or branched chain, alkyl, alkenyl, alkynyl,cyclic, heterocyclic, aromatic, or heteroaromatic.

R is N(R₁R₂), OR₁, or SR₁ where R₁ and R₂ are, independently, hydrogenor a lower alkyl group;

Y is an hydrophilic spacer sequence, or is omitted;

Z is a linking peptide, or is omitted; and

Q is a targeting moiety.

According to the present invention, the release rate of cytotoxic agentmay be controlled by modifying the chemical structure of the conjugate.For example, when R is NH₂ and n=2, the following displacement reactioncan occur:

Without wishing to be bound by any theory, in the above mechanism, thenucleophilic group aids in the intracellular release of cytotoxic ortherapeutic agent (as “X—OH”) in an adjustable fashion dependent on n.The reactivity of the nucleophile may be increased or decreasedaccording to the length of the hydrocarbon side chain, i.e., accordingto the value of n, or by changing R to NCH₃, OH, or N(CH₃)₂. Forexample, n=2 is preferable when the cytotoxic group is bound to thecarbamate linkage through an alkyl-OH group, e.g., campothecin,homocamptothecin, colchicine, combretastatin, dolistatin, doxorubicin,methotrexate, podophyllotoxin, rhizoxin, rhizixin D, rocaglamide,anguidine, a taxol, pactlitoxol, CC1065, or a maytansinoid. However,when the cytotoxic group is bound through an aryl-OH group (e.g.,combretastatin or rocaglamide) R is N(CH₃)2 and n=3 is preferable:

Thus, it is apparent from the teachings herein that conjugates of theinvention may be designed to control the release of the cytotoxic ortherapeutic agent.

The invention also features a compound having the formula:

wherein

X is a cytotoxic agent or therapeutic agent;

n is an integer from 0 to 6, wherein (CH₂)_(n) is substituted orunsubstituted, a straight or branched chain, or is an alkyl, alkenyl,alkynyl, cyclic, heterocyclic, aromatic, or heteroaromatic group;

R is N(R₁R₂), OR₁, or SR₁ where R₁ and R₂ are, independently, hydrogenor a lower alkyl group, and wherein R₃ is a (CH₂)SH group or an OHgroup. For example, a compound having the formula:

can be used to link the compound to a peptide, protein, or antibody. Inaddition, a peptide or protein can be directly synthesized onto thisconjugate, thereby providing an easy means of preparing conjugatecompounds containing a peptide or protein. The compound can be providedsuch that the R group is a tBoc protected primary or secondary aminogroup, and a peptide or protein can be added to the compound asdescribed in the examples provided below.

Peptides, proteins, and antibodies can also be attached to a compoundhaving the formula:

This unprotected R derivative can be used to link peptides, proteins,and antibodies using known thiol-reactive chemical reactions that attachto the thiol moiety at the R₃ position (see, e.g., Greg T. Hermanson,“Bioconjugate Techniques” p. 146-152, 1996, and EXAMPLE 21 below).

In all of the conjugates of the invention, X is preferably selected fromcytotoxic agents and therapeutic agents containing a primary, secondary,tertiary, benzylic or phenolic hydroxyl group. One skilled in the artwill recognize that, for those cytotoxic or therapeutic agents whichlack a hydroxyl group, a derivative containing such a hydroxyl group maybe prepared using procedures known in the art.

Preferred cytotoxic agents are those used for cancer therapy, such as,in general, alkylating agents, anti-proliferative agents, tubulinbinding agents and the like. Preferred classes of cytotoxic agentsinclude, for example, the anthracycline family of drugs, the vincadrugs, the mitomycins, the bleomycins, the cytotoxic nucleosides, thepteridine family of drugs, diynenes, and the podophyllotoxins.Particularly useful members of those classes include, for example,adriamycin, carminomycin, daunorubicin, aminopterin, methotrexate,methopterin, dichloromethotrexate, mitomycin C, porfiromycin,5-fluorouracil, 6-mercaptopurine, cytosine arabinoside, podophyllotoxin,or podophyllotoxin derivatives such as etoposide or etoposide phosphate,melphalan, vinblastine, vincristine, leurosidine, vindesine, leurosineand the like. As noted previously, one skilled in the art may makechemical modifications to the desired compound in order to makereactions of that compound more convenient for purposes of preparingconjugates of the invention. In preferred embodiments, X is a cytotoxicagent selected from camptothecin, homocamptothecin, colchicine,combretastatin, dolistatin, doxorubicin, methotrexate, podophyllotixin,rhizoxin, rhizoxin D, a taxol, paclitaxol, CC1065, or a maytansinoid,and derivatives and analogs thereof. For example, the cytotoxic agentcamptothecin is linked through its single free hydroxyl group to acarbamate linker and thiocolchicine can be derivatized such that itcontains a hydroxy group suitable for use of the described linking groupstrategy. In this embodiment of the invention, n is preferably 2 and Ris preferably NH₂.

In conjugates of the present invention, X can be any cytotoxic ortherapeutic moiety, e.g., Antineoplastic agents such as: Acivicin;Aclarubicin; Acodazole Hydrochloride; Acronine; Adozelesin; Adriamycin;Aldesleukin; Altretamine; Ambomycin; A. metantrone Acetate;Aminoglutethimide; Amsacrine; Anastrozole; Anthramycin; Asparaginase;Asperlin; Azacitidine; Azetepa; Azotomycin; Batimastat; Benzodepa;Bicalutamide; Bisantrene Hydrochloride; Bisnafide Dimesylate; Bizelesin;Bleomycin Sulfate; Brequinar Sodium;Bropirimine; Busulfan; Cactinomycin;Calusterone; Camptothecin; Caracemide; Carbetimer; Carboplatin;Carmustine; Carubicin Hydrochloride; Carzelesin; Cedefingol;Chlorambucil; Cirolemycin; Cisplatin; Cladribine; Colchicine;Combretestatin A-4; Crisnatol Mesylate; Cyclophosphamide; Cytarabine;Dacarbazine;DACA (N-[2-(Dimethyl-amino)ethyl]acridine-4-carboxamide);Dactinomycin; Daunorubicin Hydrochloride; Daunomycin; Decitabine;Dexormaplatin; Dezaguanine; Dezaguanine Mesylate; Diaziquone; Docetaxel;Dolasatins; Doxorubicin; Doxorubicin Hydrochloride; Droloxifene;Droloxifene Citrate; Dromostanolone Propionate; Duazomycin; Edatrexate;Eflornithine Hydrochloride; Ellipticine; Elsamitrucin; Enloplatin;Enpromate; Epipropidine; Epirubicin Hydrochloride; Erbulozole;Esorubicin Hydrochloride; Estramustine; Estramustine Phosphate Sodium;Etanidazole; Ethiodized Oil I 131; Etoposide; Etoposide Phosphate;Etoprine; Fadrozole Hydrochloride; Fazarabine; Fenretinide; Floxuridine;Fludarabine Phosphate; Fluorouracil; 5-FdUMP; Flurocitabine; Fosquidone;Fostriecin Sodium; Gemcitabine; Gemcitabine Hydrochloride; Gold Au 198;Homocamptothecin; Hydroxyurea; Idarubicin Hydrochloride; Ifosfamide;Ilmofosine; Interferon Alfa-2a; Interferon Alfa-2b; Interferon Alfa-n1;Interferon Alfa-n3; Interferon Beta-I a; Interferon Gamma-I b;Iproplatin; Irinotecan Hydrochloride; Lanreotide Acetate; Letrozole;Leuprolide Acetate; Liarozole Hydrochloride; Lometrexol Sodium;Lomustine; Losoxantrone Hydrochloride; Masoprocol; Maytansine;Mechlorethamine Hydrochloride; Megestrol Acetate; Melengestrol Acetate;Melphalan; Menogaril; Mercaptopurine; Methotrexate; Methotrexate Sodium;Metoprine; Meturedepa; Mitindomide; Mitocarcin; Mitocromin; Mitogillin;Mitomalcin; Mitomycin; Mitosper; Mitotane; Mitoxantrone Hydrochloride;Mycophenolic Acid; Nocodazole; Nogalamycin;Ormaplatin; Oxisuran;Paclitaxel; Pegaspargase; Peliomycin; Pentamustine; PeploycinSulfate;Perfosfamide; Pipobroman; Piposulfan; Piroxantrone Hydrochloride;Plicamycin; Plomestane; Porfimer Sodium; Porfiromycin; Prednimustine;Procarbazine Hydrochloride; Puromycin; Puromycin Hydrochloride;Pyrazofurin; Rhizoxin; Rhizoxin D; Riboprine; Rogletimide; Safingol;Safingol Hydrochloride; Semustine; Simtrazene; Sparfosate Sodium;Sparsomycin; Spirogermanium Hydrochloride; Spiromustine; Spiroplatin;Streptonigrin; Streptozocin; Strontium Chloride Sr 89; Sulofenur;Talisomycin; Taxane; Taxoid; Tecogalan Sodium; Tegafur; TeloxantroneHydrochloride; Temoporfin; Teniposide; Teroxirone; Testolactone;Thiocolchicine; Thiamiprine; Thioguanine; Thiotepa; Thymitaq;Tiazofurin; Tirapazamine; Tomudex; TOP53; Topotecan Hydrochloride;Toremifene Citrate; Trestolone Acetate; Triciribine Phosphate;Trimetrexate; Trimetrexate Glucuronate; Triptorelin; TubulozoleHydrochloride; Uracil Mustard; Uredepa; Vapreotide; Verteporfin;Vinblastine; Vinblastine Sulfate; Vincristine;Vincristine Sulfate;Vindesine; Vindesine Sulfate; Vinepidine Sulfate; Vinglycinate Sulfate;Vinleurosine Sulfate; Vinorelbine Tartrate; Vinrosidine Sulfate;Vinzolidine Sulfate; Vorozole; Zeniplatin; Zinostatin; ZorubicinHydrochloride; 2-Chlorodeoxyadenosine; 2′ Deoxyformycin;9-aminocamptothecin; raltitrexed; N-propargyl-5,8-dideazafolic acid;2chloro-2′-arabino-fluoro-2′-deoxyadenosine; 2-chloro-2′-deoxyadenosine;anisomycin; trichostatin A; hPRL-G129R; CEP-751; linomide; sulfurmustard; nitrogen mustard (mechlor ethamine); cyclophosphamide;melphalan; chlorambucil; ifosfamide; busulfan; N-methyl-Nnitrosourea(MNU); N, N′-Bis (2-chloroethyl)-N-nitrosourea (BCNU);N-(2-chloroethyl)-N′ cyclohexyl-N-nitrosourea (CCNU);N-(2-chloroethyl)-N′- (trans-4-methylcyclohexyl-Nnitrosourea (MeCCNU);N-(2-chloroethyl)-N′-(diethyl) ethylphosphonate-N-nitrosourea(fotemustine);streptozotocin; diacarbazine (DTIC); mitozolomide;temozolomide; thiotepa; mitomycin C; AZQ; adozelesin; Cisplatin;Carboplatin; Ormaplatin; Oxaliplatin;C1-973; DWA 2114R; JM216; JM335;Bis (platinum); tomudex; azacitidine; cytarabine; gemcitabine;6-Mercaptopurine; 6-Thioguanine; Hypoxanthine; teniposide 9-aminocamptothecin; Topotecan; CPT-11; Doxorubicin; Daunomycin; Epirubicin;darubicin; mitoxantrone; losoxantrone; Dactinomycin (Actinomycin D);amsacrine; pyrazoloacridine; all-trans apthal; 14-hydroxy-retro-retinol;all-trans retinoic acid; N-(4- Hydroxyphenyl)retinamide; 13-cis retinoicacid; 3-Methyl TTNEB; 9-cis retinoic acid; fludarabine (2-F-ara-AMP); or2-chlorodeoxyadenosine (2-Cda).

Other suitable anti-neoplastic compounds include, but are not limitedto, 20-pi-1,25 dihydroxyvitamin D3; 5-ethynyluracil; abiraterone;aclarubicin; acylfulvene; adecypenol; adozelesin; aldesleukin;all-tyrosine kinase antagonists; altretamine; ambamustine; amidox;amifostine; aminolevulinic acid; amrubicin; amsacrine; anagrelide;anastrozole; andrographolide; angiogenesis inhibitors; antagonist D;antagonist G; antarelix; anti-dorsalizing morphogenetic protein-1;antiandrogen, prostatic carcinoma; antiestrogen; antineoplaston;antisense oligonucleotides; aphidicolin glycinate; apoptosis genemodulators; apoptosis regulators; apurinic acid; ara-CDP-DL-PTBA;argininedeaminase; asulacrine; atamestane; atrimustine; axinastatin 1;axinastatin 2; axinastatin 3; azasetron; azatoxin; azatyrosine; baccatinIII derivatives; balanol; batimastat; BCR/ABL antagonists;benzochlorins; benzoylstaurosporine; beta lactam derivatives;beta-alethine; betaclamycin B; betulinic acid; basic fibroblast growthfactor (bFGF) inhibitor, bicalutamide; bisantrene;bisaziridinylspermine; bisnafide; bistratene A; bizelesin; breflate;bleomycin A2; bleomycin B2; bropirimine; budotitane; buthioninesulfoximine; calcipotriol; calphostin C; camptothecin derivatives (e.g., 10-hydroxy-camptothecin); canarypox IL-2; capecitabine;carboxamide-amino-triazole; carboxyamidotriazole; CaRest M3; CARN 700;cartilage derived inhibitor; carzelesin; casein kinase inhibitors(ICOS); castanospermine; cecropin B; cetrorelix; chlorins;chloroquinoxaline sulfonamide; cicaprost; cis-porphyrin; cladribine;clomifene analogues; clotrimazole; collismycin A; collismycin B;combretastatin A4; combretastatin analogue; conagenin; crambescidin 816;crisnatol; cryptophycin 8; cryptophycin A derivatives; curacin A;cyclopentanthraquinones; cycloplatam; cypemycin; cytarabine ocfosfate;cytolytic factor; cytostatin; dacliximab; decitabine; dehydrodidemnin B;2′deoxycoformycin (DCF); deslorelin; dexifosfamide; dexrazoxane;dexverapamil; diaziquone; didemnin B; didox; diethylnorspermine;dihydro-5-azacytidine; dihydrotaxol, 9-; dioxamycin; diphenylspiromustine; discodermolide; docosanol; dolasetron; doxifluridine;droloxifene; dronabinol; duocarmycin SA; ebselen; ecomustine;edelfosine; edrecolomab; eflornithine; elemene; emitefur; epirubicin;epothilones (A, R═H; B, R=Me); epithilones; epristeride; estramustineanalogue; estrogen agonists; estrogen antagonists; etanidazole;etoposide; etoposide 4′-phosphate (etopofos); exemestane; fadrozole;fazarabine; fenretinide; filgrastim; finasteride; flavopiridol;flezelastine; fluasterone; fludarabine; fluorodaunorunicinhydrochloride; forfenimex; formestane; fostriecin; fotemustine;gadolinium texaphyrin; gallium nitrate; galocitabine; ganirelix;gelatinase inhibitors; gemcitabine; glutathione inhibitors; hepsulfam;heregulin; hexamethylene bisacetamide; homoharringtonine (HHT);hypericin; ibandronic acid; idarubicin; idoxifene; idramantone;ilmofosine; ilomastat; imidazoacridones; imiquimod; immunostimulantpeptides; insulin-like growth factor-1 receptor inhibitor; interferonagonists; interferons; interleukins; iobenguane; iododoxorubicin;ipomeanol, 4-; irinotecan; iroplact; irsogladine; isobengazole;isohomohalicondrin B; itasetron; jasplakinolide; kahalalide F;lamellarin-N triacetate; lanreotide; leinamycin; lenograstim; lentinansulfate; leptolstatin; letrozole; leukemia inhibiting factor; leukocytealpha interferon; leuprolide+estrogen+progesterone; leuprorelin;levamisole; liarozole; linear polyamine analogue; lipophilicdisaccharide peptide; lipophilic platinum compounds; lissoclinamide 7;lobaplatin; lombricine; lometrexol; lonidamine; losoxantrone;lovastatin; loxoribine; lurtotecan; lutetium texaphyrin; lysofylline;lytic peptides; maytansine; mannostatin A; marimastat; masoprocol;maspin; matrilysin inhibitors; matrix metalloproteinase inhibitors;menogaril; merbarone; meterelin; methioninase; metoclopramide; MIFinhibitor; ifepristone; miltefosine; mirimostim; mismatched doublestranded RNA; mithracin; mitoguazone; mitolactol; mitomycin analogues;mitonafide; mitotoxin fibroblast growth factor-saporin; mitoxantrone;mofarotene; molgramostim; monoclonal antibody, human chorionicgonadotrophin; monophosphoryl lipid A+myobacterium cell wall sk;mopidamol; multiple drug resistance gene inhibitor; multiple tumorsuppressor 1-based therapy; mustard anticancer agent; mycaperoxide B;mycobacterial cell wall extract; myriaporone; N-acetyldinaline;N-substituted benzamides; nafarelin; nagrestip; naloxone+pentazocine;napavin; naphterpin; nartograstim; nedaplatin; nemorubicin; neridronicacid; neutral endopeptidase; nilutamide; nisamycin; nitric oxidemodulators; nitroxide antioxidant; nitrullyn; 06-benzylguanine;octreotide; okicenone; oligonucleotides; onapristone; ondansetron;ondansetron; oracin; oral cytokine inducer; ormaplatin; osaterone;oxaliplatin; oxaunomycin; paclitaxel analogues; paclitaxel derivatives;palauamine; palmitoylrhizoxin; pamidronic acid; panaxytriol; panomifene;parabactin; pazelliptine; pegaspargase; peldesine; pentosan polysulfatesodium; pentostatin; pentrozole; perflubron; perfosfamide; perillylalcohol; phenazinomycin; phenylacetate; phosphatase inhibitors;picibanil; pilocarpine hydrochloride; pirarubicin; piritrexim; placetinA; placetin B; plasminogen activator inhibitor; platinum complex;platinum compounds; platinum-triamine complex; podophyllotoxin; porfimersodium; porfiromycin; propyl bis-acridone; prostaglandin J2; proteasomeinhibitors; protein A-based immune modulator; protein kinase Cinhibitor; protein kinase C inhibitors, microalgal; protein tyrosinephosphatase inhibitors; purine nucleoside phosphorylase inhibitors;purpurins; pyrazoloacridine; pyridoxylated hemoglobin polyoxyethyleneconjugate; raf antagonists; raltitrexed; ramosetron; ras farnesylprotein transferase inhibitors; ras inhibitors; ras-GAP inhibitor,retelliptine demethylated; rhenium Re 186 etidronate; rhizoxin;ribozymes; RII retinamide; rogletimide; rohitukine; romurtide;roquinimex; rubiginone B 1; ruboxyl; safingol; saintopin; SarCNU;sarcophytol A; sargramostim; Sdi 1 mimetics; semustine; senescencederived inhibitor 1; sense oligonucleotides; signal transductioninhibitors; signal transduction modulators; single chain antigen bindingprotein; sizofiran; sobuzoxane; sodium borocaptate; sodiumphenylacetate; solverol; somatomedin binding protein; sonermin;sparfosic acid; spicamycin D; spiromustine; splenopentin; spongistatin1; squalamine; stem cell inhibitor; stem-cell division inhibitors;stipiamide; stromelysin inhibitors; sulfinosine; superactive vasoactiveintestinal peptide antagonist; suradista; suramin; swainsonine;synthetic glycosaminoglycans; tallimustine; tamoxifen methiodide;tauromustine; tazarotene; tecogalan sodium; tegafur; tellurapyrylium;telomerase inhibitors; temoporfin; temozolomide; teniposide;tetrachlorodecaoxide; tetrazomine; thaliblastine; thalidomide;thiocoraline; thrombopoietin; thrombopoietin mimetic; thymalfasin;thymopoietin receptor agonist; thymotrinan; thyroid stimulating hormone;tin ethyl etiopurpurin; tirapazamine; titanocene dichloride; topotecan;topsentin; toremifene; totipotent stem cell factor; translationinhibitors; tretinoin; triacetyluridine; triciribine; trimetrexate;triptorelin; tropisetron; turosteride; tyrosine kinase inhibitors;tyrphostins; UBC inhibitors; ubenimex; urogenital sinus-derived growthinhibitory factor; urokinase receptor antagonists; vapreotide; variolinB; vector system, erythrocyte gene therapy; velaresol; veramine;verdins; verteporfin; vinorelbine; vinxaltine; vitaxin; vorozole;zanoterone; zeniplatin; zilascorb; and zinostatin stimalamer.

X may also be an antiproliferative agent, for example piritreximisothionate. Alternatively, X may be an antiprostatic hypertrophy agentsuch as, for example, sitogluside, a benign prostatic hyperplasiatherapy agent such as, for example, tamsulosin hydrochloride, or aprostate growth inhibitor such as, for example, pentomone.

X can also be a radioactive agent, including, but not limited to:Fibrinogen ¹²⁵I; Fludeoxyglucose ¹⁸F; Fluorodopa ¹⁸F; Insulin ¹²⁵I;Insulin ¹³¹I; lobenguane ¹²³I; Iodipamide Sodium ¹³¹I; Iodoantipyrine¹³¹I; Iodocholesterol ¹³¹I; lodohippurate Sodium ¹²³I; IodohippurateSodium ¹²⁵I; Iodohippurate Sodium ¹³¹I; Iodopyracet ¹²⁵I; Iodopyracet¹³¹I; lofetamine Hydrochloride ¹²³I; Iomethin ¹²⁵; Iomethin ¹³¹;Iothalamate Sodium ¹²⁵I; Iothalamate Sodium ¹³¹I; tyrosine ¹³¹I;Liothyronine ¹²⁵I; Liothyronine ¹³¹I; Merisoprol Acetate ¹⁹⁷Hg;Merisoprol Acetate ²⁰³Hg; Merisoprol ¹⁹⁷Hg; Selenomethionine ⁷⁵Se;Technetium ^(99m)Tc Antimony Trisulfide Colloid; Technetium ^(99m)TcBicisate; Technetium ^(99m)Tc Disofenin; Technetium ^(99m)Tc Etidronate;Technetium ^(99m)Tc Exametazime; Technetium ^(99m)Tc Furifosmin;Technetium ^(99m)Tc Gluceptate; Technetium ^(99m)Tc Lidofenin;Technetium ^(99m)Tc Mebrofenin; Technetium ^(99m)Tc Medronate;Technetium ^(99m)Tc Medronate Disodium; Technetium ^(99m)Tc Mertiatide;Technetium ^(99m)Tc Oxidronate; Technetium ^(99m)Tc Pentetate;Technetium ^(99m)Tc Pentetate Calcium Trisodium; Technetium ^(99m)TcSestamibi; Technetium ^(99m)Tc Siboroxime; Technetium ^(99m)Tc;Succimer; Technetium ^(99m)Tc Sulfur Colloid; Technetium ^(99m)TcTeboroxime; Technetium ^(99m)Tc Tetrofosmin; Technetium ^(99m)TcTiatide; Thyroxine ¹²⁵I; Thyroxine ¹³¹I; Tolpovidone ¹³¹I; Triolein¹²⁵I; or Triolein ¹³¹I.

Other suitable therapeutic or cytotoxic agents used in conjugate of theinvention include, for example, anti-cancer Supplementary PotentiatingAgents, including, but not limited to: Tricyclic anti-depressant drugs(e.g., imipramine, desipramine, amitryptyline, clomipramine,trimipramine, doxepin, nortriptyline, protriptyline, amoxapine andmaprotiline); non-tricyclic anti-depressant drugs (e.g., sertraline,trazodone and citalopram); Ca++ antagonists (e.g., verapamil,nifedipine, nitrendipine and caroverine); Calmodulin inhibitors (e.g.,prenylamine, trifluoroperazine and clomipramine); Amphotericin B;Triparanol analogues (e.g., tamoxifen); antiarrhythmic drugs (e.g.,quinidine); antihypertensive drugs (e.g., reserpine); Thiol depleters(e.g., buthionine and sulfoximine) and Multiple Drug Resistance reducingagents such as Cremaphor EL.

The conjugates of the invention also can be administered with cytokinessuch as granulocyte colony stimulating factor. Preferred anticanceragents used in anti-cancer cocktails (e.g., in combination with theagents of the invention) include (some with their MTDs shown inparentheses): gemcitabine (1000 mg/m²); methotrexate (15 gm/m²i.v.+leuco.<500 mg/m² i.v. w/o leuco); 5-FU (500 mg/m²/day×5 days); FUDR(100 mg/kg×5 in mice, 0.6 mg/kg/day in human i.a.); FdUMP; Hydroxyurea(35 mg/kg/d in man); Docetaxel (60-100 mg/m²); discodermolide;epothilones; vincristine (1.4 mg/m²); vinblastine (escalating: 3.3-11.1mg/m², or rarely to 18.5 mg/m²); vinorelbine (30 mg/m²/wk); meta-pac;irinotecan (50-150 mg/m², 1 x/wk depending on patient response); SN-38(-100 times more potent than Irinotecan); 10-OH campto; topotecan (1.5mg/m²/day in humans, 1×iv LdlOmice=75 mg/m²); etoposide (100 mg/m² inman); adriamycin; flavopiridol; Cis-Pt (100 mg/m² in man); carbo-Pt (360mg/m² in man); bleomycin (20 mg/m2); mitomycin C (20 mg/m²); mithramycin(30 sug/kg); capecitabine (2.5 g/m² orally); cytarabine (100 mg/m²/day);2-Cl-2′deoxyadenosine; Fludarabine-P04 (25 mg/m²/day, ×5days);mitoxantrone (12-14 mg/m²); mitozolomide (>400 mg/m²); Pentostatin; andTomudex.

X may preferably be an antimetabolic agent, such as methotrexate.Antimetabolites include, but are not limited to, the following compoundsand their derivatives: azathioprine, cladribine, cytarabine,dacarbazine, fludarabine phosphate, fluorouracil, gencitabinechlorhydrate, mercaptopurine, methotrexate, mitobronitol, mitotane,proguanil chlorohydrate, pyrimethamine, raltitrexed, trimetrexateglucuronate, urethane, vinblastine sulfate, vincristine sulfate, etc.More preferably, X may be a folic acid-type antimetabolite, a class ofagents that includes, for example, methotrexate, proguanil chlorhydrate,pyrimethanime, trimethoprime, or trimetrexate glucuronate, orderivatives of these compounds.

In another embodiment, X can also be a member of the anthracyclinefamily of neoplastic agents, including but not limited to aclarubicinechlorhydrate, daunorubicine chlorhydrate, doxorubicine chlorhydrate,epirubicine chlorhydrate, idarubicine chlorhydrate, pirarubicine, orzorubicine chlorhydrate. Furthermore, X may be a camptothecin, or itsderivatives or related compounds such as 10, 11methylenedioxycamptothecin. X may also be selected from the maytansinoidfamily of compounds, which includes a variety of structurally relatedcompounds. For example, ansamitocin P3, maytansine,2′-N-demethylmaytanbutine, or maytanbicyclinol are maytansinoids.

Y is an hydrophilic spacer sequence that may be, preferably, a peptidethat increases the biodistribution of the conjugate, or a hydrophilicpolymer. For example, Y may be a peptide sequence that increases thehydrophilic biodistribution of the biologically active peptideconjugate. In a preferred embodiment, Y has the formula U(V-V)_(n),wherein U is D-Pro, L-Pro, D-4-OH-Pro, L-4-OH-Pro, Sarcosine, Lys, Orn,Dab, Dap, 4-NH₂-Phe, or (NH₂—(CH₂)_(m)—COOH), where m=2-10, inclusive,or is deleted; each V is independently selected from the groupconsisting of: D-Ser, L-Ser, D-Thr, L-Thr, D-Gln, L-Gln, D-Asn, L-Asn,D-4-OH-Pro, or LA hydroxy-Pro; and n=1-50, inclusive. In anotherpreferred embodiment, each V is independently D-Ser or L-Ser. In anotherpreferred embodiment, at least one V is a D-amino acid.

Following the teachings herein, Y may be selected to facilitatebiodistribution of a conjugate of the invention. Y may be a peptide or apolymer such as PEG or PVA. If Y is a hydrophilic peptide, Y maypreferably be 1 to 50 amino acids in length, or more preferably 3 to 15residues in length. For example, Y may be 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid residues in length. Whena peptide, Y may contain charged or non-polar amino acids, their analogsor derivatives that are naturally occurring, synthetic or modified.

Y can also be a hydrophilic polymer. For example, Y may be polyethyleneglycol (PEG), polyvinyl acetate, polyvinyl alcohol (PVA),N-2-hydroxypropyl) methacrylamide (HPMA) or HPMA copolymers,α,β-poly(N-hydroxyethyl)-DL-aspartamide (PHEA), orα,β-poly(N-hydroxypropyl)-DL-aspartamide. PEG, PHEA, and PVA groups usedin conjugates of the invention are also known to be excellent promotersof rapid renal secretion which is correlated generally with lowerpotential drug toxicities (see, e.g., Yamoaka et al., J. Pharmacol.Sci., 83:601-606,1994; Rypacek et al., Pflugers Arch., 392:211-217,1982; Yamoaka et al., J. Pharm. Pharmacol., 47:479-486, 1995). Thesegroups may also promote lowered toxicity emanating from bioavailable,non-internalized conjugates.

Z is a linking peptide that preserves at least fifty percent of thebiological activity of Q, when Z is bonded to Q through the terminal orside-chain amino group of Q. Generally, Z may be a peptide of 2, 3, 4,or 5 residues. Z has the formula: A-B-C-E-F, where A is D-Lys, D-Tyr,D-Ser, or L-Ser, or deleted; B is D-Lys or D-Tyr, or is deleted; C isLys, Ser, hSer, Thr, Nle, Abu, Nva, (2,3, or 4) 3-pyridyl-Ala (Pal),Orn, Dab, Dap, 4-NH₂-Phe, D-4-OH-Pro, or L-4-OH-Pro, or is deleted; E isD-Lys, D-Tyr, D-Ser, D-4-OH-Pro, L-4-OH-Pro, 3-iodo-D-Tyr, 3-5diido-D-Tyr, 3-astatine-D-Tyr, 3-5 astatine-D-Tyr, 3-bromo-D-Tyr, 3-5dibromo-D-Tyr, D-Asn, L-Asn, D-Asp, L-Asp, D-Glu, L-Glu, D-Gln, orL-Gln; and F is D-Lys, D-Tyr, D-Ser, L-Ser, D-4-OH-Pro, L-4-OH-Pro,3-iodo-D-Tyr, 3-5 diido-D-Tyr, 3-astatine-D-Tyr, 3-5 astatine-D-Tyr,3-bromo-D-Tyr, 3-5 dibromo-D-Tyr, D-Asn, L-Asn, D-Asp, L-Asp, D-Glu,L-Glu, D-Gln, or L-Gln; provided that when A, B, C, and E are Tyr, Tyr,Lys, and Tyr, respectively, F is not Lys; and when A, B, C, and E, areLys, Tyr, Lys, and Tyr, respectively, F is not Tyr or Lys; and when Aand B are deleted, and C and E are Lys and Tyr, respectively, F is notTyr or Lys.

In other embodiments, Z has the formula (when A, B, and C are deleted):

E-F

where E is D-Lys, D-Tyr, D-Ser, D-4-OH-Pro, L-4-OH-Pro, 3-iodo-D-Tyr,3-5 diiodo-D-Tyr, 3-astatine-D-Tyr, 3-5 astatine-D-Tyr, 3-bromo-D-Tyr,3-5 dibromo-D-Tyr, D-Asn, L-Asn, D-Asp, L-Asp, D-Glu, L-Glu, D-Gln, orL-Gln; and F is D-Lys, D-Tyr, D-Ser, L-Ser, D-4-OH-Pro, L-4-OH-Pro,3-iodo-D-Tyr, 3-5 diiodo-D-Tyr, 3-astatine-D-Tyr, 3-5 astatine-D-Tyr,3-bromo-D-Tyr, 3-5 dibromo-D-Tyr, D-Asn, L-Asn, D-Asp, L-Asp, D-Glu,L-Glu, D-Gln, or L-Gln.

In preferred embodiments, Z is D-Ser-Nle-D-Ser-D-Ser (SEQ ID NO: 1),D-Ser-Lys-D-Ser-D-Ser (SEQ ID NO: 2), D-Ser-Lys-D-Tyr-D-Tyr (SEQ ID NO:3), D-Ser-Lys-D-Tyr-D-Ser (SEQ ID NO: 4), D-Ser-Ser-D-Lys-D-Ser (SEQ IDNO: 5), D-Ser-Ser-D-Lys-Ser (SEQ ID NO: 5), D-Ser-Nle-D-Tyr-D-Ser (SEQID NO: 6), D-Ser-Pal-D-Tyr-D-Ser (SEQ ID NO: 7), D-Ser-Thr-D-Tyr-D-Ser(SEQ ID NO: 8), Lys-D-Ser-D-Ser (SEQ ID NO: 9), Ser-D-Lys-D-Ser (SEQ IDNO: 10), Ser-D-Lys-Ser (SEQ ID NO: 10), Nle-D-Tyr-D-Ser (SEQ ID NO: 11),Lys-D-Tyr-D-Ser (SEQ ID NO: 12), Pal-D-Lys-D-Ser (SEQ ID NO: 13),Thr-D-Tyr-D-Ser (SEQ ID NO: 14), D-Ser-D-Lys, D-Ser-D-Tyr, D-Lys-D-Lys,D-Lys-D-Tyr, or D-Tyr-D-Lys.

Q is a targeting moiety, such as a biologically active peptide.Non-immunoreactive protein, polypeptide, or peptide ligands which can beused to form the conjugates of this invention include, but are notlimited to, transferrin, epidermal growth factors (“EGF”), bombesin,gastrin, gastrin-releasing peptide, platelet-derived growth factor,IL-2, IL-6, tumor growth factors (“TGF”), such as TGF-α and TGF-β,vaccinia growth factor (“VGF”), insulin and insulin-like growth factorsI and II. Non-peptidyl ligands may include, for example, steroids,carbohydrates, vitamins, and lectins.

In preferred embodiments, Q is a peptide, such as somatostatin, asomatostatin analog, bombesin, a bombesin analog, or an antibody, suchas a monoclonal antibody. Preferred biologically active peptide ortargeting moieties are internalized by select cells via an activeinternalization process, such as when binding to a G-Protein coupledreceptor or somatostatin type 2 receptors (SSTR2).

The conjugates of the invention inhibit accumulation of toxic peptidesin tissues through inclusion of the Z group (or linking peptide (i.e.,peptide terminal extension)), containing hydrophilic residues, andoptional inclusion of an hydrophilic spacer sequence. These componentspromote rapid elimination of intact, non-bound peptides through thekidneys.

The conjugates of the invention are designed to preserve full biologicalpotency of the biologically-active peptides when conjugated to acytotoxic agent. A peptide analog of the invention has a biologicalpotency that is preferably greater than or equal to the parent peptideanalog from which it is derived, with a specificity that is greater,lesser, or equivalent to the parent peptide's target specificity. Forexample, a somatostatin analog may bind to more somatostatin receptorsubtypes than naturally-occurring somatostatin, or it may bind to aparticular receptor subtype. Some analogs of the invention containD-isomers of amino acids, or analogs thereof, facilitating stablecoupling of cytotoxic agents while retaining high receptor affinity andbiological potency of the peptide analog.

The cytotoxic or therapeutic conjugates of the invention can employ anyof the large number of known somatostatin analogs that recognize thesomatostatin receptor, such as those described above. Preferably, thesomatostatin analog portion of the conjugate contains between 10 and 18amino acids, and includes the core sequence:cyclo[Cys-Phe-D-Trp-Lys-Thr-Cys] (SEQ ID NO: 37 and SEQ ID NO: 38).Preferably, the C-terminus of the analog is: Thr-NH2. Bombesin analogs,as disclosed herein, may also be conjugated to cytotoxic or therapeuticagents in the peptide agents of the invention.

Specific targeting of therapeutic or cytotoxic agents allows selectivedestruction of a tumor expressing a receptor specific for a biologicallyactive peptide. For example, a tumor expressing a somatostatin receptorincludes a neoplasm of the lung, breast, prostate, colon, brain,gastrointestinal tract, neuroendocrine axis, liver, kidney, etc. (seeSchaer et al., Int. J. Cancer, 70:530-537, 1997; Chave et al., Br. J.Cancer 82(1):124-130, 2000; Evans et al., Br. J. Cancer 75(6):798-803,1997).

Peptides for use in conjugates of the invention include KiSS peptidesand analogs, urotensin II peptides and analogs, GnRH I and II peptidesand analogs, octreotide, depreotide, vapreotide, vasoactive intestinalpeptide (VIP), cholecystokinin (CCK), insulinlike growth factor (IGF),RGD-containing peptides, melanocyte-stimulating hormone (MSH) peptide,neurotensin, calcitonin, peptides from complementarity determiningregions of an antitumor antibody, glutathione, YIGSR (leukocyte-avidpeptides, e.g., P483H, which contains the heparin-binding region ofplatelet factor-4 (PF-4) and a lysine-rich sequence), atrial natriureticpeptide (ANP), β-amyloid peptides, delta-opioid antagonists (such asITIPP(psi)), annexin-V, endothelin, IL-1, IL-1ra, IL-2, IL-8,leukotriene B4 (LTB4), chemotactic peptides (e.g.,N-formyl-methionyl-leucyl-phenylalanine-lysine (fMLFK)), GP Iib/IIIareceptor antagonists (e.g., DMP444), epidermal growth factor, humanneutrophil elastase inhibitor (EPI-HNE-2 and EPI-HNE-4), plasmininhibitor, antimicrobial peptides, apticide (P280 and P274),thrombospondin receptor (including analogs such as TP-1300), bitistatin,pituitary adenylyl cyclase type I receptor (PAC1), fibrin α-chain,peptides derived from phage display libraries, and conservativesubstitutions thereof, that are targeted to a cell or tissue in the bodyof a mammal (e.g., diseased tissue, such as a tumor or a proliferativeangiogenic blood vessel; see, e.g., those described by Aina et al.,Biopolymers 66:184-199, 2002), and derivatives and analogs thereof. See,e.g., Signore et al., Eur. J. Nucl. Med. 28(10):1555-65, 2001.

Cytotoxic somatostatin peptide analogs may also be specific for tumorvasculatures, or angiogenic blood vessels, such as those whichover-express somatostatin receptors (see Denzler and Reubi, Cancer85:188-198, 1999; Gulec et al., J. Surg. Res. 97(2):131-137, 2001;Woltering et al., J. Surg. Res. 50:245, 1991). Additionally, because thepreferred somatostatin analogs of the invention are variouslyhydrophilic, they are water-soluble and, thus, have enhanced use ascompared to previous hydrophobic analogs. The hydrophilic analogsdescribed herein are soluble in blood, cerebrospinal fluid, and otherbodily fluids, as well as in urine, facilitating excretion by thekidneys. This hydrophilic character facilitates the delivery of theanalogs of the invention to almost every area of the body. The inventionalso discloses specific hydrophilic elements for incorporation intopeptide analogs, allowing modulation of the analog's hydrophilicity toadjust for the chemical and structural nature of the various conjugatedcytotoxic agents.

Somatostatin agonist analogs are rapidly internalized after binding totheir receptors (see, e.g., Lukinius et al, Acta Onc., 38:383-387, 1999)and can thus be used as vectors for targeting various therapeuticagents—such as traditional tumor cytotoxic agents. It is possible thatthe specificity of such anti-tumor agents can be drastically improvedsince many tumor types heavily overexpress somatostatin type 2receptors. In this manner, we propose that the toxic side effectsassociated with all conjugatable cytotoxic agents can be usefullylowered as long as a potent hybrid molecule can be designed whichretains very high affinity for somatostatin receptors.

Immunoreactive ligands for use as a targeting moiety in the inventioninclude an antigen-recognizing immunoglobulin (also referred to as“antibody”), or antigen-recognizing fragment thereof. Particularlypreferred immunoglobulins are those that can recognize atumor-associated antigen. As used herein, “immunoglobulin” refers to anyrecognized class or subclass of immunoglobulins such as IgG, IgA, IgM,IgD, or IgE. Preferred are those immunoglobulins which fall within theIgG class of immunoglobulins. The immunoglobulin can be derived from anyspecies. Preferably, however, the immunoglobulin is of human, murine, orrabbit origin. In addition, the immunoglobulin may be polyclonal ormonoclonal, but is preferably monoclonal.

Conjugates of the invention may include an antigen-recognizingimmunoglobulin fragment. Such immunoglobulin fragments may include, forexample, the Fab′, F(ab′)₂, F_(v) or Fab fragments, or otherantigen-recognizing immunoglobulin fragments. Such immunoglobulinfragments can be prepared, for example, by proteolytic enzyme digestion,for example, by pepsin or papain digestion, reductive alkylation, orrecombinant techniques. The materials and methods for preparing suchimmunoglobulin fragments are well-known to those skilled in the art. SeeParham, J. Immunology, 131, 2895, 1983; Lamoyi et al., J. ImmunologicalMethods, 56, 235, 1983.

The immunoglobulin used in conjugates of the invention may be a“chimeric antibody” as that term is recognized in the art. Also, theimmunoglobulin may be a “bifunctional” or “hybrid” antibody, that is, anantibody which may have one arm having a specificity for one antigenicsite, such as a tumor associated antigen while the other arm recognizesa different target, for example, a hapten which is, or to which isbound, an agent lethal to the antigen-bearing tumor cell. Alternatively,the bifunctional antibody may be one in which each arm has specificityfor a different epitope of a tumor associated antigen of the cell to betherapeutically or biologically modified. Hybrid antibodies thus have adual specificity, preferably with one or more binding sites specific forthe hapten of choice or one or more binding sites specific for a targetantigen, for example, an antigen associated with a tumor, an infectiousorganism, or other disease state.

Biological bifunctional antibodies are described, for example, inEuropean Patent Publication, EPA 0 105 360, which is hereby incorporatedby reference in its entirety. Such hybrid or bifunctional antibodies maybe derived either biologically, by cell fusion techniques, orchemically, such as with cross-linking agents or disulfidebridge-forming reagents, and may be comprised of whole antibodies and/orfragments thereof. Methods for obtaining such hybrid antibodies aredisclosed, for example, in PCT application W083/03679, published Oct.27, 1983, and published European Application EPA 0 217 577, publishedApr. 8, 1987, each of which is hereby incorporated by reference in itsentirety. Particularly preferred bifunctional antibodies are thosebiologically prepared from a “polydoma” or “quadroma” or which aresynthetically prepared with cross-linking agents such asbis-(maleimido)-methyl ether (“BMME”), or with other cross-linkingagents familiar to those skilled in the art

In addition, the immunoglobin may be a single chain antibody (“SCA”). AnSCA may consist of single chain Fv fragments (“scFv”) in which thevariable light (“V_(L)”) and variable heavy (“V_(H)”) domains are linkedby a peptide bridge or by disulfide bonds. Also, the immunoglobulin mayconsist of single V_(H) domains (dabs) that possess antigen-bindingactivity. See G. Winter and C. Milstein, Nature 349:295,1991; R.Glockshuber et al., Biochemistry 29:1362, 1990; and, E. S. Ward et al.,Nature 341:544, 1989.

Especially preferred for use in the present invention are chimericmonoclonal antibodies; preferably those chimeric antibodies havingspecificity toward a tumor associated antigen. As used herein, the term“chimeric antibody” refers to a monoclonal antibody comprising avariable region, i.e. a binding region, from one source or species andat least a portion of a constant region derived from a different sourceor species, usually prepared by recombinant DNA techniques. Chimericantibodies having a murine variable region and a human constant regionare especially preferred in certain applications of the invention,particularly human therapy, because such antibodies are readily preparedand may be less immunogenic than purely murine monoclonal antibodies.Such murine/human chimeric antibodies are the product of expressedimmunoglobulin genes comprising DNA segments encoding murineimmunoglobulin variable regions and DNA segments encoding humanimmunoglobulin constant regions. Other forms of chimeric antibodies foruse in conjugates of the invention are those in which the class orsubclass has been modified or changed from that of the originalantibody. Such “chimeric” antibodies are also referred to as“class-switched antibodies.” Methods for producing chimeric antibodiesinvolve conventional recombinant DNA and gene transfection techniquesnow well known in the art. See Morrison, S. L, et al., Proc. Nat'l Acad.Sci., 81:6851, 1984.

The term “chimeric antibody” also includes a “humanized antibody,”namely, those antibodies in which the framework or “complementaritydetermining regions” (“CDR”) have been modified to include the CDR of animmunoglobulin of different specificity, as compared to that of theparent immunoglobulin. In a preferred embodiment, a murine CDR isgrafted into the framework region of a human antibody to prepare the“humanized antibody.” See, e.g., L. Riechmann et al., Nature 332:323,1988; M. S. Neuberger et al., Nature 314:268, 1985. Particularlypreferred CDRs correspond to those representing sequences recognizingthe antigens noted above for the chimeric and bifunctional antibodies.See, e.g., EPA 0 239 400 (published Sep. 30, 1987), which is herebyincorporated by reference in its entirety.

One skilled in the art will recognize that a bifunctional-chimericantibody can be prepared which would have the benefits of lowerimmunogenicity of the chimeric or humanized antibody, as well as theflexibility, especially for therapeutic treatment, of the bifunctionalantibodies described above. Such bifunctional-chimeric antibodies can besynthesized, for instance, by chemical synthesis using cross-linkingagents and/or recombinant methods of the type described above. Thepresent invention should not be construed as limited in scope by anyparticular method of production of an antibody whether bifunctional,chimeric, bifunctional-chimeric, humanized, or an antigen-recognizingfragment or derivative thereof.

Conjugates of the invention may also include immunoglobulins (as definedabove) or immunoglobulin fragments to which are fused active proteins,for example, an enzyme of the type disclosed in Neuberger, et al., PCTapplication W086/01533, published Mar. 13, 1986, which is herebyincorporated by reference in its entirety.

As used herein, “bifunctional,” “fused,” “chimeric” (includinghumanized), and “bifunctional-chimeric” (including humanized) antibodyconstructions also include, within their individual contexts,constructions including antigen recognizing fragments. As one skilled inthe art will recognize, such fragments may be prepared by traditionalenzymatic cleavage of intact bifunctional, chimeric, humanized, orchimeric-bifunctional antibodies. In the event that intact antibodiesare not susceptible to such cleavage, e.g., because of the nature of theconstruction involved, the noted constructions can be prepared withimmunoglobulin fragments used as the starting materials; or, ifrecombinant techniques are used, the DNA sequences, themselves, can betailored to encode the desired “fragment” which, when expressed, can becombined in vivo or in vitro, by chemical or biological means, toprepare the final desired intact immunoglobulin “fragment.” It is inthis context that the term “fragment” is used herein.

The immunoglobulin (antibody), or fragment thereof, used in conjugatesof the present invention may be polyclonal or monoclonal in nature.Monoclonal antibodies are the preferred immunoglobulins. The preparationof polyclonal or monoclonal antibodies is well known to those skilled inthe art. See, e.g., G. Kohler and C. Milstein, Nature 256:495, 1975. Inaddition, hybridomas and/or monoclonal antibodies which are produced bysuch hybridomas and which are useful in the practice of the presentinvention are publicly available.

In a further embodiment, the invention features a method for thetreatment of a disease or modification of a biological function. Themethod includes administering to a warm-blooded animal in need thereof,a therapeutically effective or biological function modifying amount of aconjugate of the invention. One of skill in the art will recognize thatthe particular conjugate used will depend on the disease state to betreated or the biological system to be modified. In particular, oneskilled in the art will be able to select a particular targeting moietyand cytotoxic or therapeutic agent to prepare a conjugate of theinvention which has specificity for the treatment of the disease or isable to modify the biological function desired. It is envisioned thatseveral disease are treatable using the conjugates of the invention,including, for example, tumors of the lung, breast, brain, eye, prostateor colon; tumors of neuroendocrine origin (e.g., carcinoid syndrome);and proliferative angiogenic blood vessels (in, e.g., the eye), such asthose associated with tumors, retinal macular degeneration, or diabeticretinopathy.

Conjugates of the invention may be administered to a mammalian subject,such as a human, directly or in combination with any pharmaceuticallyacceptable carrier or salt known in the art. Pharmaceutically acceptablesalts may include non-toxic acid addition salts or metal complexes thatare commonly used in the pharmaceutical industry. Examples of acidaddition salts include organic acids such as acetic, lactic, pamoic,maleic, citric, malic, ascorbic, succinic, benzoic, palmitic, suberic,salicylic, tartaric, methanesulfonic, toluenesulfonic, ortrifluoroacetic acids or the like; polymeric acids such as tannic acid,carboxymethyl cellulose, or the like; and inorganic acids such ashydrochloric acid, hydrobromic acid, sulfuric acid phosphoric acid, orthe like. Metal complexes include zinc, iron, and the like. Oneexemplary pharmaceutically acceptable carrier is physiological saline.Other physiologically acceptable carriers and their formulations areknown to one skilled in the art and described, for example, inRemington's Pharmaceutical Sciences, (18^(th) edition), ed. A. Gennaro,1990, Mack Publishing Company, Easton, Pa.

Pharmaceutical formulations of a therapeutically effective amount of aconjugate of the invention, or pharmaceutically acceptable salt-thereof,can be administered orally, parenterally (e.g., intramuscular,intraperitoneal, intravenous, subcutaneous, or ocular injection,inhalation, intradermally, optical drops, or implant), nasally,vaginally, rectally, sublingually or topically, in admixture with apharmaceutically acceptable carrier adapted for the route ofadministration.

Methods well known in the art for making formulations are found, forexample, in Remington's Pharmaceutical Sciences (18^(th) edition), ed.A. Gennaro, 1990, Mack Publishing Company, Easton, Pa. Compositionsintended for oral use may be prepared in solid or liquid forms accordingto any method known to the art for the manufacture of pharmaceuticalcompositions. The compositions may optionally contain sweetening,flavoring, coloring, perfuming, and/or preserving agents in order toprovide a more palatable preparation. Solid dosage forms for oraladministration include capsules, tablets, pills, powders, and granules.In such solid forms, the active compound is admixed with at least oneinert pharmaceutically acceptable carrier or excipient. These mayinclude, for example, inert diluents, such as calcium carbonate, sodiumcarbonate, lactose, sucrose, starch, calcium phosphate, sodiumphosphate, or kaolin. Binding agents, buffering agents, and/orlubricating agents (e.g., magnesium stearate) may also be used. Tabletsand pills can additionally be prepared with enteric coatings.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, solutions, suspensions, syrups, and soft gelatincapsules. These forms contain inert diluents commonly used in the art,such as water or an oil medium. Besides such inert diluents,compositions can also include adjuvants, such as wetting agents,emulsifying agents, and suspending agents.

Formulations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, or emulsions. Examples of suitablevehicles include propylene glycol, polyethylene glycol, vegetable oils,gelatin, hydrogenated naphalenes, and injectable organic esters, such asethyl oleate. Such formulations may also contain adjuvants, such aspreserving, wetting, emulsifying, and dispersing agents. Biocompatible,biodegradable lactide polymer, lactide/glycolide copolymer, orpolyoxyethylene-polyoxypropylene copolymers may be used to control therelease of the compounds. Other potentially useful parenteral deliverysystems for the polypeptides of the invention include ethylene-vinylacetate copolymer particles, osmotic pumps, implantable infusionsystems, and liposomes.

Liquid formulations can be sterilized by, for example, filtrationthrough a bacteria-retaining filter, by incorporating sterilizing agentsinto the compositions, or by irradiating or heating the compositions.Alternatively, formulations can also be manufactured in the form ofsterile, solid compositions that can be dissolved in sterile water orsome other sterile injectable medium immediately before use.

Compositions for rectal or vaginal administration are preferablysuppositories that may contain, in addition to active substances,excipients such as cocoa butter or a suppository wax. Compositions fornasal or sublingual administration are also prepared with standardexcipients known in the art. Formulations for inhalation may containexcipients, for example, lactose, or may be aqueous solutionscontaining, for example, polyoxyethylene-9-lauryl ether, glycocholateand deoxycholate, or may be oily solutions for administration in theform of nasal drops or spray, or as a gel.

The amount of active ingredient in the compositions of the invention canbe varied. One skilled in the art will appreciate that the exactindividual dosages may be adjusted somewhat depending upon a variety offactors, including the polypeptide being administered, the time ofadministration, the route of administration, the nature of theformulation, the rate of excretion, the nature of the subject'sconditions, and the age, weight, health, and gender of the patient Inaddition, the severity of the condition targeted by the biologicallyactive peptide such as somatostatin or bombesin will also have an impacton the dosage level. Generally, dosage levels of between 0.1 μg/kg to100 mg/kg of body weight are administered daily as a single dose ordivided into multiple doses. Preferably, the general dosage range isbetween 250 μg/kg to 5.0 mg/kg of body weight per day. Wide variationsin the needed dosage are to be expected in view of the differingefficiencies of the various routes of administration. For instance, oraladministration generally would be expected to require higher dosagelevels than administration by intravenous injection. Variations in thesedosage levels can be adjusted using standard empirical routines foroptimization, which are well known in the art. In general, the precisetherapeutically effective dosage will be determined by the attendingphysician in consideration of the above identified factors.

The conjugates of the invention can be administered in a sustainedrelease composition, such as those described in, for example, U.S. Pat.No. 5,672,659 and U.S. Pat. No. 5,595,760. The use of immediate orsustained release compositions depends on the type of condition beingtreated. If the condition consists of an acute or over-acute disorder, atreatment with an immediate release form will be preferred over aprolonged release composition. Alternatively, for preventative orlong-term treatments, a sustained released composition will generally bepreferred.

Polypeptides used in the present invention can be prepared in anysuitable manner. The polypeptides may be isolated from naturallyoccurring sources, recombinantly produced, or produced synthetically, orproduced by a combination of these methods. The synthesis of shortpeptides is well known in the art. See e.g. Stewart et al., Solid PhasePeptide Synthesis (Pierce Chemical Co., 2d ed., 1984). The peptides ofthe present invention can be synthesized according to standard peptidesynthesis methods known in the art.

The following examples are meant to illustrate the invention. They arenot meant to limit the invention in any way.

EXAMPLE 1 Preparation of the chloroformate of camptothecin

Camptothecin (250 mg) and 4-dimethylaminopyridine (DMAP; 50 mg) weresuspended in 3 mL anhydrous pyridine and 50 mL anhydrous methylenechloride. Phosgene (750 μL of a 20% solution in toluene) was added tothe slurry and mixed 2 h at ambient temperature. Excess phosgene andmethylene chloride were evaporated in a chemical fume hood and thechlorformate of camptothecin dissolved in DCM.

EXAMPLE 2 Preparation ofCamptothecin-carbonyl-N-aminoethyl-glycine-D-tert-butyl-Ser-Nle-D-tert-butyl-Tyr-D-tert-butyl-Ser-S-trityl-Cys-Phe-D-Trp-epsilon-tert-butyloxycarbonyl-Lys-tert-butyl-Thr-S-trityl-Cys-tert-butyl-Thr-Rink-amide-resin(SEQ ID NO: 16)

Rink amide[4-(2′,4′-dimethoxyphenyl-Fmoc-(aminomethyl)phenoxyacetamido-norleucyl-methylbenzhydrylamineresin (0.063 mmole), 100-200 mesh (Novabiochem, San Diego, Calif.) wasadded to the reaction vessel of a CS136 automatic peptide synthesizer(CS Bio, Inc., San Carlos, Calif.) and swollen in dimethylformamide(DMF) for approximately 1 hour. The resin was filtered and an excess of20% piperidine in DMP was added and mixed (2 min). The resin wasfiltered and again an excess amount of 20% piperidine added and mixed(20 min) to ensure complete removal of the resin Fmoc group. Afterdeprotection, the resin was washed 4 times with DMF and then the firstprotected amino acid, Fmoc-Thr(tBut) (0.188 mmol),diisopropylcarbodiimide (DIC) (0.188 mmol), and N-hydroxybenzotriazolemonohydrate (HOBt) (0.188 mmol) were all dissolved in DMF and added tothe resin, which was mixed for 1 h, followed by washing 4 times withDMF.

The Fmoc group was again removed by treatment with 20% piperidine/DMFsolution and, following the same general coupling procedures, thefollowing amino acids were successively reacted with the growing peptidechain: Fmoc-S-trityl-L-cysteine, Fmoc-O-t-butyl-L-threonine,N^(α)-Fmoc-N^(ε)-Boc-L-lysine, N^(α)-Fmoc-N^(in)-Boc-D-tryptophan,Fmoc-L-phenylalanine, Fmoc-S-trityl-L-cysteine, Fmoc-O-t-butyl-D-serine,Fmoc-O-t-butyl-D-tyrosine, N^(α)-Fmoc-Norleucine,Fmoc-O-t-butyl-D-serine, bromoacetic acid. After completion ofbromoacetic acid coupling to peptidyl resin (3 eq),N-Boc-ethylenediamine was added in N-methyl-α-pyrrolidinone (NMP) andmixed (2 h) and then washed 3 times with DMF and 3 times with DCM.Camptothecin chloroformate from EXAMPLE 1 was added to the resin andmixed overnight, washed with copious amounts of DMF followed bymethanol. After a final filtration the derivatized resin was air driedovernight.

EXAMPLE 3 Preparation ofcamptothecin-carbonyl-N-(2-aminoethyl)-glycine-D-Ser-Nle-D-Tyr-D-Ser-cyclo[Cys-Phe-D-Trp-Lys-Thr-Cys]-Thr-amide(SEQ ID NO: 17)

The camptothecin-peptide resin prepared in EXAMPLE 2 (0.063 mmol) wasplaced in a round bottomed flask to which was added 15 mL of a solutionof trifluoroacetic acid (TFA) containing water (2.5%), 1,2-ethanedithiol(2.5%), and triisopropylsilane (1%). The suspension was agitated (2 h)and filtered and washed several times with TFA. The TFA was evaporatedin vacuo and ether added to the resulting oil to give a yellow powderthat was then dissolved in 60% acetic acid (250 mL). A concentratedsolution of iodine in methanol was added dropwise with vigorous stirringuntil a permanent brown coloration was formed whereupon excess iodinewas removed by addition of a small quantity of ascorbic acid.

The solution was reduced to a volume of around 10 mL in vacuo and thecrude camptothecin peptide purified by preparative reverse phase highpressure liquid chromatography (RP-HPLC) on a column (21.4×250 mm) ofC-18 bonded silica (Dynamax 300, 8 μm). A linear gradient elution systemat a flow rat of 20 mL/min was employed: buffer A consisted of 0.1% TFAand buffer B, 0.1% TFA in 80% MeCN; 20% B to 50% B was increased at 1%per min. The separation was monitored at 280 nm. The fractionscontaining the pure product, as identified by analytical HPLC, werepooled, concentrated in vacuo, and subjected to lyophilization. Thepeptide was obtained as a fluffy yellow powder of constant weight bylyophilization from aqueous acetic acid. Correct composition wasdemonstrated by amino acid analysis of an acid hydrolysate and matrixassisted laser desorption mass spectrometry.

EXAMPLE 4 Preparation of chloroformate of paclitaxel

Paclitaxel (0.6 mmole) was dissolved in 30 mL anhydrous DCM in a 100 mLround bottomed (RB) flask. To this solution, diisopropylethylamine(DIEA; 3 mmol) dissolved in 20 mL anhydrous DCM was added over 20minutes at 0° C. under a dry nitrogen atmosphere. Phosgene (3 mmol) wasadded to the slurry and mixed for 30 minutes at 0° C. and 2 hours atambient temperature. Excess phosgene and methylene chloride wereevaporated and the chloroformate of paclitaxel was dissolved inmethylene chloride.

EXAMPLE 5 Preparation ofPaclitaxel-carbonyl-N-(2-hydroxyethyl)-glycine-D-trityl-Ser-Norleucine-D-trityl-Tyr-D-trityl-Ser-S-trityl-Cys-Phe-D-Trp-epsilon-mtt-Lys-trityl-Thr-S-trityl-Cys-trityl-Thr-Amino-xanthen-MBHA-resin (SEQ ID NO: 16)

FMOC-Amino-Xanthen-MBHA (4-Methylbenzhydrylamine Hydrochloride)polystyrene resin (0.063 mmol) [Bachem, catalog # D-2040, Lot # 0541173]was added to the reaction vessel of a CS136 automatic peptidesynthesizer (CS Bio, Inc., San Carlos, Calif.) and swollen in DMF forapproximately 1 hour. The resin was filtered and an excess of 20%piperidine in DMF was added and mixed for 2 minutes. The resin wasfiltered and again an excess amount of 20% piperidine added and mixedfor 20 minutes to ensure complete removal of the resin Fmoc group. Afterdeprotection, the resin was washed 4 times with DMF and then the firstprotected amino acid, Fmoc-Thr(trityl) (0.188 mmol),diisopropylcarbodiimide (DIC) (0.188 mmol), and N-hydroxybenzotriazolemonohydrate (HOBt) (0.188 mmol) were all dissolved in DMF and added tothe resin which was mixed (1 h) followed by washing 4 times with DMF.

The Fmoc group was again removed by treatment with 20% piperidine/DMFsolution and, following the same general coupling procedures, thefollowing amino acids were successively reacted with the growing peptidechain: Fmoc-S-trityl-L-cysteine, Fmoc-O-trityl-L-threonine,N^(α)-Fmoc-N^(ε)mtt-L-lysine, N^(α)-Fmoc-D-tryptophan,Fmoc-L-phenylalanine, Fmoc-S-trityl-L-cysteine, Fmoc-O-trityl-D-serine,Fmoc-O-trityl-D-tyrosine, N-Fmoc-Norleucine, Fmoc-O-trityl-D-serine,Bromoacetic acid. After completion of bromoacetic acid coupling topeptidyl resin, (3 eq) ethanolamine was added in NMP and mixed for twohours then washed 3 times with DMF and 3 times with DCM. Paclitaxelchloroformate from EXAMPLE 4 was added to the resin and mixed overnight,washed with copious amounts of DMF followed by methanol. After a finalfiltration the derivatized resin was air dried overnight.

EXAMPLE 6 Preparation ofPaclitaxel-carbonyl-N-(2-hydroxyethyl)-glycine-D-Ser-Me-D-Tyr-D-Ser-cyclo[Cys-Phe-D-Trp-Lys-Thr-Cys]-Thr-amide(SEQ ID NO: 17)

The paclitaxel-peptide resin prepared in EXAMPLE 5 (0.063 mmol) wasplaced in a RB flask to which was added 15 mL of a solution oftrifluoroacetic acid (2%) in methylene chloride. The suspension wasagitated (2 h), filtered, and washed several times with methylenechloride. The methylene chloride was evaporated in vacuo and ether addedto the resulting oil to give a white powder that was then dissolved in60% acetic acid (250 mL). A concentrated solution of iodine in methanolwas added dropwise with vigorous stirring until a permanent browncoloration was formed whereupon excess iodine was removed by addition ofa small quantity of ascorbic acid.

The solution was reduced to a volume of around 10 mL in vacuo and thecrude paclitaxel peptide purified by preparative reverse phase highpressure liquid chromatography (RP-HPLC) on a column (21.4×250 mm) ofC-18 bonded silica (Dynamax 300, 8 μm). A linear gradient elution systemat a flow rat of 20 mL/min was employed: buffer A consisted of 0.1% TFAand buffer B, 0.1% TFA in 80% MeCN; 20% B to 50% B was increased at 1%per min. The separation was monitored at 280 mu. The fractionscontaining the pure product as evidenced by analytical HPLC were pooled,concentrated in vacuo, and subjected to lyophilization. The peptide wasobtained as a fluffy white powder of constant weight by lyophilizationfrom aqueous acetic acid. Correct composition was demonstrated by aminoacid analysis of an acid hydrolysate and matrix assisted laserdesorption mass spectrometry.

EXAMPLE 7 Preparation ofCamptothecin-carbonyl-N-aminoethyl-glycine-D-tert-butyl-Ser-D-epsilon-tert-butyloxycarbonyl-Lys-Gln-Trp-Ala-Val-β-Ala-trityl-His-Phe-Nle-Rink-amide-resin(SEQ ID NO: 18)

Rink amide MBHA polystyrene resin (0.063 mmol)[4-(2′,4′-dimethoxyphenyl-Fmoc-(aminomethyl)phenoxyacetamido-norleucyl-methylbenzhydrylamineresin, 100-200 mesh, Novabiochem, San Diego, Calif.] was added to thereaction vessel of a CS136 automatic peptide synthesizer (CS Bio, Inc.,San Carlos, Calif.) and swollen in DMF for approximately 1 hour. Theresin was filtered and an excess of 20% piperidine in DMF was added andmixed for 2 minutes. The resin was filtered and again an excess amountof 20% piperidine added and mixed for 20 minutes to ensure completeremoval of the resin Fmoc group. After deprotection, the resin waswashed 4 times with DMF and then the first protected amino acid,Fmoc-Norleucine) (0.188 mmol), diisopropylcarbodiimide (DIC) (0.188mmol), and N-hydroxybenzotriazole monohydrate (HOBt) (0.188 mmol) wereall dissolved in DMF and added to the resin which was mixed (1 h)followed by washing 4 times with DMF.

The Fmoc group was again removed by treatment with 20% piperidine/DMFsolution and, following the same general coupling procedures, thefollowing amino acids were successively reacted with the growing peptidechain: Fmoc-L-phenylalanine, Fmoc-N^(im)-trityl-L-histidine, Fmoc-βAla,Fmoc-Valine, Fmoc-alanine, Fmoc-N^(in)-Boc-L-tryptophan, Fmoc-glutamine,Fmoc-N^(g)-Boc-D-lysine, Fmoc-O-t-butyl-D-serine, bromoacetic acid.After completion of bromoacetic acid coupling to peptidyl resin, (3 eq)N-Boc-ethylenediamine was added in NMP and mixed for two hours thenwashed 3 times with DMF and 3 times with DCM. Camptothecin chloroformatefrom EXAMPLE 1 was added to the resin and mixed overnight, washed withcopious amounts of DMF followed by methanol. After a final filtrationthe derivatized resin was air dried overnight.

EXAMPLE 8 Preparation ofCamptothecin-carbonyl-N-(2-aminoethyl)-glycine-D-Ser-D-Lys-Gln-Trp-Ala-Val-β-Ala-His-Phe-Me-amide(SEQ ID NO: 18)

The camptothecin-peptide resin prepared in EXAMPLE 7 (0.063 mmol) wasplaced in a round bottomed flask to which was added 15 mL of a solutionof trifluoroacetic acid (TFA) containing water (2.5%), andtriisopropylsilane (1%). The suspension was agitated (2 h), filtered,and washed several times with TFA. The TFA was evaporated in vacuo andether added to the resulting oil to give a yellow powder that was thendissolved in 60% acetic acid (250 mL).

The solution was reduced to a volume of around 10 mL in vacuo and thecrude camptothecin peptide purified by preparative reverse phase highpressure liquid chromatography (RP-HPLC) on a column (21.4×250 mm) ofC-18 bonded silica (Dynamax 300, 8 μm). A linear gradient elution systemat a flow rat of 20 mL/min was employed: buffer A consisted of 0.1% TFAand buffer B, 0.1% TFA in 80% MeCN; 20% B to 50% B was increased at 1%per min. The separation was monitored at 280 nm. The fractionscontaining the pure product as evidenced by analytical HPLC were pooled,concentrated in vacuo, and subjected to lyophilization. The peptide wasobtained as a fluffy yellow powder of constant weight by lyophilizationfrom aqueous acetic acid. Correct composition was demonstrated by aminoacid analysis of an acid hydrolysate and matrix assisted laserdesorption mass spectrometry.

EXAMPLE 9 Preparation ofCamptothecin-carbonyl-N-aminoethyl-glycine-D-tert-butyl-Ser-D-tert-butyl-Tyr-Gln-Trp-Ala-Val-β-Ala-trityl-His-Phe-Nle-Rink-amide-resin(SEQ ID NO: 19)

Rink amide MBHA polystyrene resin (0.063 mmol)[4-(2′,4′-dimethoxyphenyl-Fmoc-(aminomethyl)phenoxyacetamido-norleucyl-methylbenzhydrylamineresin, 100-200 mesh, Novabiochem, San Diego, Calif.] was added to thereaction vessel of a CS136 automatic peptide synthesizer (CS Bio, Inc.,San Carlos, Calif.) and swollen in DMF for approximately 1 hour. Theresin was filtered and an excess of 20% piperidine in DMF was added andmixed for 2 minutes. The resin was filtered and again an excess amountof 20% piperidine added and mixed for 20 minutes to ensure completeremoval of the resin Fmoc group. After deprotection, the resin waswashed 4 times with DMF and then the first protected amino acid,Fmoc-Norleucine (0.188 mmol), diisopropylcarbodiimide (DIC) (0.188mmol), and N-hydroxybenzotriazole monohydrate (HOBt) (0.188 mmol) wereall dissolved in DMF and added to the resin which was mixed (1 h)followed by washing 4 times with DMF.

The Fmoc group was again removed by treatment with 20% piperidine/DMFsolution and, following the same general coupling procedures, thefollowing amino acids were successively reacted with the growing peptidechain: Fmoc-L-phenylalanine, Fmoc-N^(im)-trityl-L-histidine, Fmoc-βAla,Fmoc-Valine, Fmoc-alanine, Fmoc-N^(in)-Boc-L-tryptophan, Fmoc-glutamine,Fmoc-O-t-butyl-D-Tyr, Fmoc-O-t-butyl-D-serine, bromoacetic acid. Aftercompletion of bromoacetic acid coupling to peptidyl resin, (3 eq)N-Boc-ethylenediamine was added in NMP and mixed for two hours thenwashed 3 times with DMF and 3 times with DCM. Camptothecin chloroformatefrom EXAMPLE 1 was added to the resin and mixed overnight, washed withcopious amounts of DMF followed by methanol. After a final filtrationthe derivatized resin was air dried overnight.

EXAMPLE 10 Preparation ofCamptothecin-carbonyl-N-(2-aminoethyl)-glycine-D-Ser-D-Tyr-Gln-Trp-Ala-Val-β-Ala-His-Phe-Nle-amide(SEQ D NO: 19)

The camptothecin-peptide resin prepared in EXAMPLE 9 (0.063 mmol) wasplaced in a RB flask to which was added 15 mL of a solution oftrifluoroacetic acid (TFA) containing water (2.5%), andtriisopropylsilane (1%). The suspension was agitated (2 h), filtered andwashed several times with TFA. The TFA was evaporated in vacuo and etheradded to the resulting oil to give a yellow powder that was thendissolved in 60% acetic acid (250 mL).

The solution was reduced to a volume of around 10 mL in vacuo and thecrude camptothecin peptide purified by preparative reverse phase highpressure liquid chromatography (RP-HPLC) on a column (21.4×250 mm) ofC-18 bonded silica (Dynamax 300, 8 μm). A linear gradient elution systemat a flow rat of 20 mL/min was employed: buffer A consisted of 0.1% TFAand buffer B, 0.1% TFA in 80% MeCN; 20% B to 50% B was increased at 1%per min. The separation was monitored at 280 run. The fractionscontaining the pure product as evidenced by analytical HPLC were pooled,concentrated in vacuo, and subjected to lyophilization. The peptide wasobtained as a fluffy yellow powder of constant weight by lyophilizationfrom aqueous acetic acid. Correct composition was demonstrated by aminoacid analysis of an acid hydrolysate and matrix assisted laserdesorption mass spectrometry.

EXAMPLE 11 Preparation ofCombretastatin-carbonyl-N-(2-aminoethyl)-glycine-D-tert-butyl-Ser-Norleucine-D-tert-butyl-Tyr-D-tert-butyl-Ser-S-trityl-Cys-Phe-D-Trp-epsilon-tert-butyloxycarbonyl-Lys-tert-butyl-Thr-S-trityl-Cys-tert-butyl-Thr-Rink-amide-resin (SEQ IDNO: 39)

Rink amide MBHA polystyrene resin (0.063 mmole)[4-(2′,4′-dimethoxyphenyl-Fmoc-(aminomethyl)phenoxyacetamido-norleucyl-methylbenzhydrylamineresin, 100-200 mesh, Novabiochem, San Diego, Calif.] was added to thereaction vessel of a CS136 automatic peptide synthesizer (CS Bio, Inc.,San Carlos, Calif.) and swollen in DMF for approximately 1 hour. Theresin was filtered and an excess of 20% piperidine in DMF was added andmixed for 2 minutes. The resin was filtered and again an excess amountof 20% piperidine added and mixed for 20 minutes to ensure completeremoval of the resin Fmoc group. After deprotection, the resin waswashed 4 times with DMF and then the first protected amino acid,Fmoc-Thr(tBut) (0.188 mmol), diisopropylcarbodiimide (DIC) (0.188 mmol),and N-hydroxybenzotriazole monohydrate (HOBt) (0.188 mmol) were alldissolved in DMF and added to the resin which was mixed (1 h) followedby washing 4 times with DMF.

The Fmoc group was again removed by treatment with 20% piperidine/DMFsolution and, following the same general coupling procedures, thefollowing amino acids were successively reacted with the growing peptidechain: Fmoc-S-trityl-L-cysteine, Fmoc-O-t-butyl-L-threonine,N^(α)-Fmoc-N-Boc-L-lysine, N^(a)-Fmoc-N^(in)-Boc-D-tryptophan,Fmoc-L-phenylalanine, Fmoc-S-trityl-L-cysteine, Fmoc-O-t-butyl-D-serine,Fmoc-O-t-butyl-D-tyrosine, N^(α)-Fmoc-Norleucine,Fmoc-O-t-butyl-D-serine, Bromoacetic acid. After completion ofbromoacetic acid coupling to peptidyl resin, (3 eq)N-Boc-ethylenediamine was added in NMP and mixed for two hours thenwashed 3 times with DMF and 3 times with DCM. Combretastatinchloroformate from EXAMPLE 1 was added to the resin and mixed overnight,washed with copious amounts of DMF followed by methanol. After a finalfiltration the derivatized resin was air dried overnight

EXAMPLE 12 Preparation ofcombretastatin-carbonyl-N-(2-aminoethyl)-glycine-D-Ser-Me-D-Tyr-D-Ser-cyclo[Cys-Phe-D-Trp-Lys-Thr-Cys]-Thr-amide(SEQ ID NO: 40)

The combretastatin-peptide resin prepared in Example 11 (0.063 mmol) wasplaced in a round bottomed flask to which was added 15 mL of a solutionof trifluoroacetic acid (TFA) containing water (2.5%), 1,2-ethanedithiol(2.5%), and trisopropylsilane (1%). The suspension was agitated (2 h),filtered and washed several times with TFA. The TFA was evaporated invacuo and ether added to the resulting oil to give a white powder thatwas then dissolved in 60% acetic acid (250 mL). A concentrated solutionof iodine in methanol was added dropwise with vigorous stirring until apermanent brown coloration was formed whereupon excess iodine wasremoved by addition of a small quantity of ascorbic acid.

The solution was reduced to a volume of around 10 mL in vacuo and thecrude camptothecin peptide purified by preparative reverse phase highpressure liquid chromatography (RP-HPLC) on a column (21.4×250 mm) ofC-18 bonded silica (Dynamax 300, 8 μm). A linear gradient elution systemat a flow rat of 20 mL/min was employed: buffer A consisted of 0.1% TFAand buffer B, 0.1% TFA in 80% MeCN; 20% B to 50% B was increased at 1%per min. The separation was monitored at 280 nm. The fractionscontaining the pure product as evidenced by analytical HPLC were pooled,concentrated in vacuo and subjected to lyophilization. The peptide wasobtained as a fluffy white powder of constant weight by lyophilizationfrom aqueous acetic acid. Correct composition was demonstrated by aminoacid analysis of an acid hydrolysate and matrix assisted laserdesorption mass spectrometry.

EXAMPLE 13 Preparation ofCamptothecin-carbonyl-N-(N-methyl-2-aminoethyl)-glycine-D-tert-butyl-Ser-Norleucine-D-tert-butyl-Tyr-D-tert-butyl-Ser-S-trityl-Cys-Phe-D-Trp-epsilon-tert-butyloxycarbonyl-Lys-tert-butyl-Thr-S-trityl-Cys-tert-butyl-Thr-Rink-amide-resin(SEQ ID NO: 39)

Rink amide MBHA polystyrene resin (0.063 mmol)[4-(2′,4′-dimethoxyphenyl-Fmoc-(aminomethyl)phenoxyacetamido-norleucyl-methylbenzhydrylamineresin, 100-200 mesh, Novabiochem, San Diego, Calif.] was added to thereaction vessel of a CS136 automatic peptide synthesizer (CS Bio, Inc.,San Carlos, Calif.) and swollen in DMF for approximately 1 hour. Theresin was filtered and an excess of 20% piperidine in DMF was added andmixed for 2 minutes. The resin was filtered and again an excess amountof 20% piperidine added and mixed for 20 minutes to ensure completeremoval of the resin Fmoc group. After deprotection, the resin waswashed 4 times with DMF and then the first protected amino acid,Fmoc-Thr(tBut) (0.188 mmol), diisopropylcarbodiimide (DIC) (0.188 mmol),and N-hydroxybenzotriazole monohydrate (HOBt) (0.188 mmol) were alldissolved in DMF and added to the resin which was mixed (1 h) followedby washing 4 times with DMF.

The Fmoc group was again removed by treatment with 20% piperidine/DMFsolution and, following the same general coupling procedures, thefollowing amino acids were successively reacted with the growing peptidechain: Fmoc-S-trityl-L-cysteine, Fmoc-O-t-butyl-L-threonine,N^(α)-Fmoc-N⁶⁸-Boc-L-lysine, N^(α)-Fmoc-N^(in)-Boc-D-tryptophan,Fmoc-L-phenylalanine, Fmoc-S-trityl-L-cysteine, Fmoc-O-t-butyl-D-serine,Fmoc-O-t-butyl-D-tyrosine, N^(α)-Fmoc-Norleucine,Fmoc-O-t-butyl-D-serine, Bromoacetic acid. After completion ofbromoacetic acid coupling to peptidyl resin, (3 eq)N-Boc-N-methylethylenediamine was added in NMP and mixed for two hoursthen washed 3 times with DMF and 3 times with DCM. Camptothecinchloroformate from EXAMPLE 1 was added to the resin and mixed overnight,washed with copious amounts of DMF followed by methanol. After a finalfiltration the derivatized resin was air dried overnight

EXAMPLE 14 Preparation ofcamptothecin-carbonyl-N-(N-methyl-2-aminoethyl)-glycine-D-Ser-Nle-D-Tyr-D-Ser-cyclo[Cys-Phe-D-Trp-Lys-Thr-Cys]-Thr-amide(SEQ ID NO: 40)

The camptothecin-peptide resin prepared in EXAMPLE 13 (0.063 mmol) wasplaced in a RB flask to which was added 15 mL of a solution oftrifluoroacetic acid (TFA) containing water (2.5%), 1,2-ethanedithiol(2.5%), and triisopropylsilane (1%). The suspension was agitated (2 h),filtered and washed several times with TFA. The TFA was evaporated invacuo and ether added to the resulting oil to give a yellow powder thatwas then dissolved in 60% acetic acid (250 mL). A concentrated solutionof iodine in methanol was added dropwise with vigorous stirring until apermanent brown coloration was formed whereupon excess iodine wasremoved by addition of a small quantity of ascorbic acid.

The solution was reduced to a volume of around 10 mL in vacuo and thecrude camptothecin peptide purified by preparative reverse phase highpressure liquid chromatography (RP-HPLC) on a column (21.4×250 mm) ofC-18 bonded silica (Dynamax 300, 8 μm). A linear gradient elution systemat a flow rat of 20 mL/min was employed: buffer A consisted of 0.1% TFAand buffer B, 0.1% TFA in 80% MeCN; 20% B to 50% B was increased at 1%per min. The separation was monitored at 280 nm. The fractionscontaining the pure product as evidenced by analytical HPLC were pooled,concentrated in vacuo and subjected to lyophilization. The peptide wasobtained as a fluffy yellow powder of constant weight by lyophilizationfrom aqueous acetic acid. Correct composition was demonstrated by aminoacid analysis of an acid hydrolysate and matrix assisted laserdesorption mass spectrometry.

EXAMPLE 15di-tert-butyloxycarbonyl-His-Leu-Gln-Ile-Gln-Pro-tert-butyloxycarbonyl-Trp-tert-butyl-Tyr-Pro-Gln-Ile-tert-butyl-Ser-N-e-camptothecin-carbonyl-N-(N-2-aminoethyl)-glycine-Lys-tert-butyl-Ser-Rink-amide-resin(SEQ ID NO: 20)

Rink amide MBHA polystyrene resin (0.063 mmol) resin, 100-200 mesh,Novabiochem, San Diego, Calif.) was added to the reaction vessel of aCS136 automatic peptide synthesizer (CS Bio, Inc., San Carlos, Calif.)and swollen in DMF for approximately 1 hour. The resin was filtered andan excess of 20% piperidine in DMF was added and mixed for 2 minutes.The resin was filtered and again an excess amount of 20% piperidineadded and mixed for 20 minutes to ensure complete removal of the resinFmoc group. After deprotection, the resin was washed 4 times with DMFand then the first protected amino acid, Fmoc-Serine(tBut) (0.188 mmol),diisopropylcarbodiimide (DIC) (0.188 mmol), and N-hydroxybenzotriazolemonohydrate (HOBt) (0.188 mmol) were all dissolved in DMF and added tothe resin which was mixed (1 h) followed by washing 4 times with DMF.

The Fmoc group was again removed by treatment with 20% piperidine/DMFsolution and, following the same general coupling procedures, thefollowing amino acids were successively reacted with the growing peptidechain:Fmoc-N-e-1(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)3-3methyl-butyl-L-Lysine,Fmoc-O-t-butyl-L-Serine, N^(α)-Fmoc—L-Isoleucine, N-Fmoc-L-Glutamine,N-Fmoc-L-Proline, N-Fmoc-O-Trityl-L-Tyrosine,N^(α)-Fmoc-N^(in)-Boc-L-tryptophan, N-Fmoc-L-Proline,N-Fmoc-L-Glutamine, N^(α)-Fmoc—L-Isoleucine, N-Fmoc-L-Glutamine,N^(α)-Fmoc-Leucine, Boc-O-t-butyl-L-Histidine.

The protected peptide resin was then treated 3 times (3 min each) with a2% solution of hydrazine hydrate in DMF in order to remove theN-e-1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)3-methyl-butyl (ivDde)group on the lysine residue. The free e-amino group was then coupledwith bromoacetic acid followed by addition of N-Boc-N-ethylenediamine (3eq, 12 h) in NMP. Camptothecin chloroformate from EXAMPLE 1 was added tothe resin and mixed overnight, washed with copious amounts of DMFfollowed by methanol. After a final filtration the derivatized resin wasair dried overnight.

EXAMPLE 16 Preparation of Camptothecin Derivative of Phage Peptide p147:His-Leu-Gln-Ile-Gln-Pro-Trp-Tyr-Pro-Gin-Ile-Ser-N-e-camptothecin-carbonyl-N-(N-2-aminoethyl)-glycine-Lys-Ser-NH₂(SEQ ID NO: 20)

The camptothecin-peptide resin prepared in EXAMPLE 15 (0.063 mmol) wasplaced in a RB flask to which was added 15 mL of a solution oftrifluoroacetic acid (TFA) containing water (2.5%), 1,2-ethanedithiol(2.5%), and triisopropylsilane (1%). The suspension was agitated (5 h),filtered and washed several times with TFA. The TFA was evaporated invacuo and ether added to the resulting oil to give a yellow powder whichwas purified by preparative reverse phase high pressure liquidchromatography (RP-HPLC) on a column (21.4×250 mm) of C-18 bonded silica(Dynamax 300, 8 μm). A linear gradient elution system at a flow rat of20 mL/min was employed: buffer A consisted of 0.1% TFA and buffer B,0.1% TFA in 80% MeCN; 20% B to 50% B was increased at 1% per min. Theseparation was monitored at 280 nm. The fractions containing the pureproduct as evidenced by analytical HPLC were pooled, concentrated invacua, and subjected to lyophilization. The peptide was obtained as afluffy yellow powder of constant weight by lyophilization from aqueousacetic acid. Correct composition was demonstrated by amino acid analysisof an acid hydrolysate and matrix assisted laser desorption massspectrometry.

EXAMPLE 17 Preparation of N-carboxyanhydride of O-t-butyl-D,L-serine

O-t-butyl-D-serine (0.0062 mol) and O-t-butyl-L-serine (0.0062 mol) weresuspended in 55 mL anhydrous tetrahydrofuran (THF) under nitrogenatmosphere. Phosgene (0.025 mol) was added to suspension and thesuspension was refluxed for 15 minutes. Solvent and excess phosgene wereevaporated and the resulting oil was dissolved in 10 mL THF then addedto 600 mL Hexanes and crystallized at −20° C.

EXAMPLE 18 Preparation ofCamptothecin-carbonyl-N-(2-aminoethyl)-glycine-[D or LO-tert-butyl-Ser]₋₁₅-D-tert-butyl-Ser-Norleucine-D-tert-butyl-Tyr-D-tert-butyl-Ser-S-trityl-Cys-Phe-D-Trp-epsilon-tert-butyloxycarbonyl-Lys-tert-butyl-Thr-S-trityl-Cys-tert-butyl-Thr-Rink-amide-resin (SEQ ID NO: 21, and SEQID NO: 23-SEQ ID NO 36)

Rink amide MBHA polystyrene resin (0.063 mmol)[4-(2′,4′-dimethoxyphenyl-Fmoc-(aminomethyl)phenoxyacetamido-norleucyl-methylbenzhydrylamineresin, 100-200 mesh, Novabiochem, San Diego, Calif.] was added to thereaction vessel of a CS136 automatic peptide synthesizer (CS Bio, Inc.,San Carlos, Calif.) and swollen in DMF for approximately 1 hour. Theresin was filtered and an excess of 20% piperidine in DMF was added andmixed for 2 minutes. The resin was filtered and again an excess amountof 20% piperidine added and mixed for 20 minutes to ensure completeremoval of the resin Fmoc group. After deprotection, the resin waswashed 4 times with DMF and then the first protected amino acid,Fmoc-Thr(tBut) (0.188 mmol), diisopropylcarbodiimide (DIC) (0.188 mmol),and N-hydroxybenzotriazole monohydrate (HOBt) (0.188 mmol) were alldissolved in DMF and added to the resin which was mixed (1 h) followedby washing 4 times with DMF.

The Fmoc group was again removed by treatment with 20% piperidine/DMFsolution and, following the same general coupling procedures, thefollowing amino acids were successively reacted with the growing peptidechain: Fmoc-S-trityl-L-cysteine, Fmoc-O-t-butyl-L-threonine,N^(α)-Fmoc-N^(ε)-Boc-L-lysine, N^(α)-Fmoc-N^(in)-Boc-D-tryptophan,Fmoc-L-phenylalanine, Fmoc-S-trityl-L-cysteine, Fmoc-O-t-butyl-D-serine,Fmoc-O-t-butyl-D-tyrosine, N^(α)-Fmoc-Norleucine,Fmoc-O-t-butyl-D-serine. At this point the N-terminal Fmoc group wasremoved, the resin washed several times with DMF, then the resin wastransferred to a 100 mL RB flask. O-tert-butyl-Serine NCA (1.5 g) wasdissolved in anhydrous DMF and added to the peptidyl resin and thesuspened resin was shaken overnight at 40° C. The resin was filtered andwashed several times with DMF then placed back on the CS Biosynthesizer. Bromoacetic acid was then coupled to the N-terminal serinein DCM/DIC. After completion of Bromoacetic acid coupling to peptidylresin, (3 eq) N-Boc-ethylenediamine was added in NMP and mixed for twohours then washed 3 times with DMF and 3 times with DCM. Camptothecinchloroformate from EXAMPLE 1 was added to the resin and mixed overnight,washed with copious amounts of DMF followed by methanol. After a finalfiltration the derivatized resin was air dried overnight

EXAMPLE 19 Preparation ofcamptothecin-carbonyl-N-(2-aminoethyl)-glycine-[Poly-D,L-Ser]-D-Ser-Nle-D-Tyr-D-Ser-cyclo[Cys-Phe-D-Trp-Lys-Thr-Cys]-Thr-amide(SEQ ID NO: 22)

The camptothecin-peptide resin prepared in EXAMPLE 18 (0.063 mmol) wasplaced in a RB flask to which was added 15 mL of a solution oftrifluoroacetic acid (TFA) containing water (2.5%), 1,2-ethanedithiol(2.5%), and triisopropylsilane (1%). The suspension was agitated (2 h),filtered, and washed several times with TFA. The TFA was evaporated invacuo and ether added to the resulting oil to give a yellow powder thatwas then dissolved in 60% acetic acid (250 mL). A concentrated solutionof iodine in methanol was added dropwise with vigorous stirring until apermanent brown coloration was formed whereupon excess iodine wasremoved by addition of a small quantity of ascorbic acid.

The solution was reduced to a volume of around 10 mL in vacuo and thecrude camptothecin peptide purified by preparative reverse phase highpressure liquid chromatography (RP-HPLC) on a column (21.4×250 mm) ofC-18 bonded silica (Dynamax 300, 8 μm). A linear gradient elution systemat a flow rate of 20 mL/min was employed: buffer A consisted of 0.1% TFAand buffer B, 0.1% TFA in 80% MeCN; 20% B to 50% B was increased at 1%per min. The separation was monitored at 280 nm. The fractionscontaining the pure product as evidenced by analytical HPLC were pooled,concentrated in vacuo, and subjected to lyophilization. The peptide wasobtained as a fluffy yellow powder of constant weight by lyophilizationfrom aqueous acetic acid. Correct composition was demonstrated by aminoacid analysis of an acid hydrolysate and matrix assisted laserdesorption mass spectrometry.

EXAMPLE 20 In Vivo and In Vitro Use of Conjugate Carbamate Compounds

Conjugate compounds with a carbamate linker were tested in vitro and invivo for release rate of the cytotoxic agent. A carbamate linker waschosen because it is reasonably stable in plasma compared, for example,to commonly used ester-type linkages. We also tested carbamate compoundscontaining an intramolecular cyclic moiety that allows adjustment of therelease rate of the alcoholic component of the carbamate culminating inthe release of an attached alcohol derivative and formation of a cyclicurea which can be utilized to control release rates of suitablecytotoxins (FIG. 1). It is known that the pKa value of the leavingalcohol of a carbamate is important for the stability of the prodrug,therefore, we chose two different chemically suitable cytotoxic agentseach containing one hydroxyl group with signicantly different pKavalues. One cytotoxic agent was the topoisomerase I inhibitor,camptothecin, which bears a tertiary hydroxyl group in ring position 20(FIG. 1) with a pKa of approximately 18. The other cytotoxic agent wasthe tubulin-binding agent, combretastatin, which has one phenolichydroxyl group attached to an aromatic ring, thus exhibiting a muchlower pKa value of approximately 10.3.

All peptide conjugates shown in Table 1 were synthesized totally on Rinkamide resin support using the standard FMOC (9-fluorenylmethoxycarbonyl)protection/deprotection strategy employing simple DIC couplings and HBTUactivation as a second step only if a Kaiser test was positive (asdescribed above). Once the peptide portion of the conjugate wascomplete, bromoacetic acid was coupled to the N-terminus using DIC/DCM.After washing the resin, a 5 M excess of N-BOC-ethylenediamine (or theappropriate protected diamine) in NUT was added and mixed for 1 h.Seperately, camptothecin (200 mg; Aldrich) and DMAP (400 mg) inanhydrous DCM (40 mL) were mixed and cooled to 0° C. To this suspensionwas added 20% phosgene in toluene (600 uL) and the mixture was allowedto react for about 45 min. After solvent evaporation, the powder wassuspended in DCM (40 mL) and added to the peptidyl resin bearing thefree secondary amine and allowed to react overnight followed by washingseveral times with DMF, DCM, and methanol. The peptide was then cleavedfrom the resin for 2 h using the standard acid mixture TFA:H₂0:EDT:TIS,95:2:2:1. After cleavage, the conjugate was precipitated 4 times inethyl ether, dissolved in 60% HOAC, and cyclized with iodine inmethanol. It was then subjected to preparative chromatography andcharacterized by MS and amino acid analysis. All peptides were obtainedin at least 90+% purity and yields were around 35% theoretical. Fullretention of conjugate agonists potencies relative to somatostatinitself was demonstrated by their ability to inhibit gonadotropinreleasing hormone (GNRH)-stimulated GH release from monolayer culturesof rat pituitary cells, an assay system that has been shown to correlatewell with binding affnity to the human type 2 somatostatin receptor(see, e.g., Raynor et al., Mol. Pharmacol. 43:838, 1993). Inhibitorypotencies (IC50's) of the conjugates shown in Table 2 ranged from 0.26to 0.49 nM compared to 0.63 nM for somatostatin-14 itself. Thisretention of full affinity is due to the employment of the N-terminalNle-d-Tyr-d-Ser tripeptide extension of the cyclic somaostatin portionof the conjugate that we have found generally allows large groups to beattached with little to no loss of affinity. TABLE 1 Structures ofcamptothecin and combretastatin conjugates containing various BINARlinking groups.

R₁ R₂ Compound

1 2 3 4 5

6

TABLE 2 Agonist activity, cytotoxicity, and buffer and serum half-lifeof compounds 1-6 and control compounds Half Life Growth CytotoxicityPhosphate Half-Life Hormone IC₅₀, nM^(a) Buffer Rat Serum InhibitionCompounds IMR-32 Cells (Hours) (Hours) IC₅₀, nM^(a) 1  374 ± 18Stable^(a) 106 0.32 ± 0.02 2 54.2 ± 6 123 18 0.27 ± 0.02 3  571 ± 48Stable 59 0.30 ± 0.04 4 >1000 Stable 72 0.26 ± 0.03 5 >1000 na na 0.49 ±0.1 6 2.79 ± 0.33  31 4 0.31 ± 0.1 Camptothecin 2.21 ± 0.44 na na naCombretastatin   4.36 na na na SRIF (1-14) na^(b) na 2-5 0.63 ± 0.029minutes^(a)Experiment carried out for 50 h.^(b)Somtostatin analogues were non-toxic to IMR32 cells at up to 10⁻⁵ Mconcentrations.

Each peptide conjugate was subjected to phosphate buffer and rat serumstability studies. The analogues were incubated in either 0.1 Mphosphate buffer or fresh rat serum at 37° C. and aliquots were taken atdifferent time points and examined by HPLC. Compounds 1 ,3 , and 4showed complete buffer stability for the length of the experiment (50 h;Table 2). Compound 2 was less stable in buffer with a half-life ofaround 120 h, while compound 6 was the least stable with a half-life ofonly 30 h in buffer. In rat serum, this same trend was evident, exceptthat there was clearly a catabolic effect of serum on the stability ofthe compounds. Compounds 1, 3, and 4 were again the most stable, whilecompound 2's half-life was 18 h and combretastatin compound 6, whichcontains the phenolic hydroxyl, was least stable with a 4 h half-life.

The cytotoxic activities of these conjugates were measured using astandard MTT assay kit (Promega Corporation, Madison, Wis.). Eachcompound was incubated for 3 days with human neuroblastoma IMR32 cells(somatostatin receptor overexpression) at different concentrations inquadruplate and IC50's were generated from the dose-response curves(FIG. 2). Although the combretastatin-ethlylenediamine BINAR compound 6was the most cytotoxic, after reviewing the stability data in buffer, itis evident that 50% of this conjugate is free cytoxin after 3 days andperhaps more since cell media will contain some enzymes. TheN-Me-ethylenediamine camptothecin conjugate 2 was the second mostpotent. After 3 days, more than 80% of this conjugate was still intactThe next most potent was the ethlylenediamine compound 1. This analoguedemonstrated complete stability in phosphate buffer, but was seven timesless potent than compound 2 and slightly more active than compound 3even though it was more stable in serum. This property is likely due tocompound 1's ability to form a cyclic urea while compound 3 is unable toform this same species.

In preliminary tumor bearing animal studies, compounds 1 and 2 appear tobe equally effective at inducing a cytotoxic effect in the tumor afterrepeated intraperitoneal administration. Compounds based onN,N-dimethyethylenediamine, diaminopropyl, and 2-hydroxyethylamine BINARgroups (FIG. 2; Table 2) were essentially inactive presumably due tolack of release of any free cytotoxic agent intra- or extracellularly.The lead camptothecin conjugate 2 was able to significantly stabilizethe growth of transplanted NCI-H69 small cell lung carcinomas(expression of SSTR2) in nude mice at doses far below the maximumtolerated equivalent doses of camptothecin alone. The data suggest notonly a chemical component to the release rate of the alcohol but alsosome enzymatic component.

The value of the BINAR linking strategy lies both in its ability to beadjusted to accommodate ideal release rates for various types of alcoholcontaining groups and its ease of incorporation onto a peptide freeamino group by simple solid-phase chemistries. The chemistry forintroducing the groups is well suited for solid phase synthesis and iseasily adapted for many different peptides, therapeutic agents, andcytotoxins. Although this strategy is readily applied to the N-terminus,it can also be applied to an orthogonally protected side chain as wellshould this be necessary for preservation of peptide binding affinity.With the present camptothecin conjugates, high cytotoxicity was achievedwith compounds 1 and 2 (FIG. 2; Table 2) even though they were verystable to cell media incubation conditions. High concentrations ofintact peptide conjugate will be specifically bound to tumor cellswhereupon internalization would take place. Furthermore, the conjugatesare highly soluble and tissue clearance rates and routes can be readilyfurther adjusted by altering the hydrophilicity of the peptidecomponent, as described above. If the peptide conjugate remainsreasonably intact during clearance, then toxic side effects should bemuch reduced.

EXAMPLE 21 Preparation ofCamptothecin-carbonyl-N-aminoethyl-glycine-S-trityl-Cys-Rink-amide-resin

Rink amide[4-(2′,4′-dimethoxyphenyl-Fmoc-(aminomethyl)phenoxyacetamido-norleucyl-methylbenzhydrylamineresin (0.063 mmole), 100-200 mesh (Novabiochem, San Diego, Calif.) wasadded to the reaction vessel of a CS136 automatic peptide synthesizer(CS Bio, Inc., San Carlos, Calif.) and swollen in dimethylformamide(DMF) for approximately 1 h. The resin was filtered and an excess of 20%piperidine in DMF was added and mixed (2 min). The resin was filteredand again an excess amount of 20% piperidine added and mixed (20 min) toensure complete removal of the resin Fmoc group. After deprotection, theresin was washed 4 times with DMF and then the first protected aminoacid, Fmoc-S-trityl-L-cysteine (0.188 mmol), diisopropylcarbodiimide(DIC) (0.188 mmol), and N-hydroxybenzotriazole monohydrate (HOBt) (0.188mmol) were all dissolved in DMF/DCM and added to the resin, which wasmixed for 1 h, followed by washing 4 times with DMF.

The Fmoc group was again removed by treatment with 20% piperidine/DMFsolution and, following the same general coupling procedures,Fmoc-N-(2-Boc-aminoethyl)glycine (Neosystem) was added. The Fmoc groupwas removed and the peptidyl resin washed several times with DCM.Camptothecin chloroformate from EXAMPLE 1 was added to the resin andmixed overnight, then washed with copious amounts of DMF followed bymethanol. After a final filtration the derivatized resin was air driedovernight.

Preparation of camptothecin-carbonyl-N-(2-aminoethyl)-glycine-Cys-amide

The camptothecin-peptide resin prepared in EXAMPLE 21 (0.063 mmol) wasplaced in a round bottomed flask to which was added 15 mL of a solutionof trifluoroacetic acid (TFA) containing water (2.5%), 1,2-ethanedithiol(2.5%), and triisopropylsilane (1%). The suspension was agitated (2 h),filtered, and washed several times with TFA. The TFA was evaporated invacuo and ether added to the resulting oil to give a yellow powder thatwas then dissolved in 60% acetic acid (15 mL).

The crude camptothecin peptide was purified by preparative reverse phasehigh pressure liquid chromatography (RP-HPLC) on a column (21.4×250 mm)of C-18 bonded silica (Dynamax 300, 8 μm). A linear gradient elutionsystem at a flow rat of 20 mL/min was employed: buffer A consisted of0.1% TFA and buffer B, 0.1% TFA in 80% MeCN; 20% B to 50% B wasincreased at 1% per min. The separation was monitored at 280 nm. Thefractions containing the pure product, as identified by analytical HPLC,were pooled, concentrated in vacuo, and subjected to lyophilization. Thepeptide was obtained as a fluffy yellow powder of constant weight bylyophilization from aqueous acetic acid. Correct composition, as shownbelow, was demonstrated by matrix assisted laser desorption massspectrometry.

EXAMPLE 23 Preparation ofCamptothecin-carbonyl-N-aminoethyl-glycine-Phe(benzophenone)-amide-resin

Rink amide[4-(2′,4′-dimethoxyphenyl-Fmoc-(aminomethyl)phenoxyacetamido-norleucyl-methylbenzhydrylamineresin (0.063 mmole), 100-200 mesh (Novabiochem, San Diego, Calif.) wasadded to the reaction vessel of a CS136 automatic peptide synthesizer(CS Bio, Inc., San Carlos, Calif.) and swollen in dimethylformamide(DMF) for approximately 1 h. The resin was filtered and an excess of 20%piperidine in DMF was added and mixed (2 min). The resin was filteredand again an excess amount of 20% piperidine added and mixed (20 min) toensure complete removal of the resin Fmoc group. After deprotection, theresin was washed 4 times with DMF and then the first protected aminoacid, Fmoc-L-p-benzoyl-Phenylalanine (0.188 mmol),diisopropylcarbodiimide (DIC) (0.188 mmol), and N-hydroxybenzotriazolemonohydrate (HOBt) (0.188 mmol) were all dissolved in DMF/DCM and addedto the resin, which was mixed for 1 h, followed by washing 4 times withDMF.

The Fmoc group was again removed by treatment with 20% piperidine/DMFsolution and, following the same general coupling procedures,Fmoc-N-(2-Boc-aminoethyl)glycine (Neosystem) was added. The Fmoc groupwas removed and the peptidyl resin washed several times with DCM.Camptothecin chloroformate from EXAMPLE 1 was added to the resin andmixed overnight, then washed with copious amounts of DMF followed bymethanol. After a final filtration the derivatized resin was air driedovernight.

EXAMPLE 24 Preparation ofcamptothecin-carbonyl-N-(2-aminoethyl)-glycine-p-Benzoyl-phenylalanine-amide

The camptothecin-peptide resin prepared in EXAMPLE 23 (0.063 mmol) wasplaced in a round bottomed flask to which was added 15 mL of a solutionof trifluoroacetic acid (TFA) containing water (2.5%), andtriisopropylsilane (1%). The suspension was agitated for 2 h, filtered,and washed several times with TFA. The TFA was evaporated in vacuo andether added to the resulting oil to give a yellow powder that was thendissolved in 60% acetic acid (15 mL).

The crude camptothecin peptide was purified by preparative reverse phasehigh pressure liquid chromatography (RP-HPLC) on a column (21.4×250 mm)of C-18 bonded silica (Dynamax 300, 8 μm). A linear gradient elutionsystem at a flow rat of 20 mL/min was employed: buffer A consisted of0.1% TFA and buffer B, 0.1% TFA in 80% MECN; 20% B to 50% B wasincreased at 1% per min. The separation was monitored at 280 nm. Thefractions containing the pure product, as identified by analytical HPLC,were pooled, concentrated in vacuo, and subjected to lyophilization. Thepeptide was obtained as a fluffy yellow powder of constant weight bylyophilization from aqueous acetic acid. Correct composition, as shownbelow, was demonstrated by matrix assisted laser desorption massspectrometry.

Other Embodiments

Although the present invention has been described with reference topreferred embodiments, one skilled in the art can easily ascertain itsessential characteristics and without departing from the spirit andscope thereof, can make various changes and modifications of theinvention to adapt it to various usages and conditions. Those skilled inthe art will recognize or be able to ascertain using no more thanroutine experimentation, many equivalents to the specific embodiments ofthe invention described herein. Such equivalents are intended to beencompassed in the scope of the present invention.

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindependent publication or patent application was specifically andindividually indicated to be incorporated by reference.

1. A conjugate compound represented by the following formula:

wherein: X is a cytotoxic agent or therapeutic agent; n is an integerfrom 0 to 6, wherein (CH₂)_(n) is substituted or unsubstituted, astraight or branched chain, or is an alkyl, alkenyl, alkynyl, cyclic,heterocyclic, aromatic, or heteroaromatic group; R is N(R₁R₂), OR₁, orSR₁, wherein R₁ and R₂ are, independently, hydrogen or a lower alkylgroup; Y is hydrophilic spacer sequence, or is omitted; Z is a linkingpeptide that, when bonded to Q at the N-terminus or at a compatibleside-chain amino group of Q, preserves at least 50% of the biologicalactivity of Q, Z having the formula: A-B-C-E-F, wherein: A is D-Lys,D-Tyr, D-Ser, or L-Ser, or is deleted; B is D-Lys or D-Tyr, or isdeleted; C is Lys, Ser, hSer, Thr, Nle, Abu, Nva, (2, 3, or 4)3-pyridyl-Ala (Pal), Orn, Dab, Dap, 4-NH₂-Phe, D-4-OH-Pro, orLA4-OH-Pro, or is deleted; E is D-Lys, D-Tyr, D-Ser, D-4-OH-Pro,L-4-OH-Pro, 3-iodo-D-Tyr, 3-5 diiodo-D-Tyr, 3-astatine-D-Tyr, 3-5astatine-D-Tyr, 3-bromo-D-Tyr, 3-5 dibromo-D-Tyr, D-Asn, L-Asn, D-Asp,L-Asp, D-Glu, L-Glu, D-Gln, or L-Gln; and F is D-Lys, D-Tyr, D-Ser,L-Ser, D-4-OH-Pro, L-4-OH-Pro, 3-iodo-D-Tyr, 3-5 diiodo-D-Tyr,3-astatine-D-Tyr, 3-5 astatine-D-Tyr, 3-bromo-D-Tyr, 3-5 dibromo-D-Tyr,D-Asn, L-Asn, D-Asp, L-Asp, D-Glu, L-Glu, D-Gln, or L-Gln; provided thatwhen A, B, C, and E are Tyr, Tyr, Lys, and Tyr, respectively, F is notLys; and when A, B, C, and B are Lys, Tyr, Lys, and Tyr, respectively, Eis not Tyr or Lys; and when A and B are deleted, and C and E are Lys andTyr, respectively, F is not Tyr or Lys; and Q is a targeting moiety, oris omitted.
 2. The compound of claim 1, wherein X is a cytotoxic agent.3. The compound of claim 2, wherein said cytotoxic agent is analkylating agent, an antibiotic, an antimetabolite, a tubulin inhibitor,a topoisomerase I or II inhibitor, an hormonal agonist or antagonists,an apoptotic agent, or an immunomodulator.
 4. The compound of claim 2,wherein said cytotoxic agent is camptothecin, homocamptothecin,colchicine, combretastatin, dolistatin, doxorubicin, methotrexate,podophyllotoxin, rhizoxin, rhizoxin D, a taxol, paclitaxol, CC1065, amaytansinoid, or derivatives or analogs thereof.
 5. The compound ofclaim 1, wherein said hydrophilic spacer sequence is a peptide thatincreases the hydrophilic biodistribution of said compound.
 6. Thecompound of claim 5, wherein said peptide has the formula U(V-V)_(n),wherein U is D-Pro, L-Pro, D-4-OH-Pro, L-4-OH-Pro, Sarcosine, Lys, Orn,Dab, Dap, 4-NH₂-Phe, or (NH₂—(CH₂)_(m)—COOH), where m=2-10, inclusive,or is deleted; each V is independently selected from the groupconsisting of: D-Ser, L-Ser, D-Thr, L-Thr, D-Gln, L-Gln, D-Asn, L-Asn,D-4-OH-Pro, or LA hydroxy-Pro; and n=1-50, inclusive.
 7. The compound ofclaim 6, wherein V is independently D-Ser or L-Ser.
 8. The compound ofclaim 6, wherein at least one V is a D-amino acid.
 9. The compound ofclaim 1, wherein said hydrophilic spacer sequence is a hydrophilicpolymer.
 10. The compound of claim 9, wherein said hydrophilic polymeris polyethylene glycol, polyvinyl acetate, polyvinyl alcohol, HPMA(N-(2-hydroxypropyl) methacrylamide) or HPMA copolymers, α,β-poly(N-hydroxyethyl)-DL-aspartamide (PHEA), or α,β-poly(N-hydroxypropyl)-DL-aspartamide.
 11. The compound of claim 1,wherein said targeting moiety is a biologically-active peptide.
 12. Thecompound of claim 11, wherein said biologically-active peptide issomatostatin, bombesin, a KiSS peptide, a urotensin II peptide,gonadotropin-releasing hormone (GnRH) I and II peptides, octreotide,depreotide, vapreotide, vasoactive intestinal peptide (VIP),cholecystokinin (CCK), insulin-like growth factor (IGF), RGD-containingpeptides, melanocyte-stimulating hormone (MSH) peptide, neurotensin,calcitonin, a peptide comprising the complementarity determining regionof an antitumor antibody, glutathione, a leukocyte-avid peptidecomprising the amino acid sequence YIGSR, the heparin-binding region ofplatelet factor-4 (PF-4), and a lysine-rich sequence, atrial natriureticpeptide (ANP), a β-amyloid peptide, a delta-opioid antagonist,annexin-V, endothelin, interleukin (IL)-1, IL-1ra, IL-2, IL-8,leukotriene B4 (LTB4), a chemotactic peptide, a GP IIb/IIIa receptorantagonist, epidermal growth factor, a human neutrophil elastaseinhibitor, plasmin inhibitor, an antimicrobial peptide, apticide P280,apticide P274, a thrombospondin receptor, bitistatin, pituitary adenylylcyclase type I receptor (PAC1), fibrin α-chain, or derivatives oranalogs thereof.
 13. The compound of claim 11, wherein saidbiologically-active peptide is an antibody or an antibody fragment 14.The compound of claim 12, wherein said leukocyte-avid peptide is P483H.15. The compound of claim 12, wherein said delta-opioid antagonist isITIPP(psi).
 16. The compound of claim 12, wherein said chemotacticpeptide is N-formyl-methionyl-leucyl-phenylalanine-lysine (fMLFK). 17.The compound of claim 12, wherein said GP IIb/IIIa receptor antagonistis DMP444.
 18. The compound of claim 12, wherein said human neutrophilelastase inhibitor is EPI-HNE-2 or EPI-HNE-4.
 19. The compound of claim12, wherein said thrombospondin receptor is TP-1300.
 20. The compound ofclaim 11, wherein said biologically-active peptide targets said compoundto a cell or tissue in the body of a mammal.
 21. The compound of claim1, wherein said targeting moiety is a peptide derived from aphage-display library, or conservative substitutions thereof, thattargets said compound to a cell or tissue in the body of a mammal. 22.The compound of claim 20 or 21, wherein said cell or tissue comprises acancer cell, a white blood cell, cardiac tissue, brain tissue, or atubercle infected with tuberculosis.
 23. The compound of claim 20 or 21,wherein said tissue is a tumor or a proliferative angiogenic bloodvessel.
 24. The compound of claim 23, wherein said blood vessel is inthe eye.
 25. The compound of claim 1, wherein Q is a somatostatinpeptide.
 26. The compound of claim 1, wherein Q is a bombesin peptide.27. The compound of claim 13, wherein said antibody is a monoclonalantibody or a fragment thereof.
 28. The compound of claim 1, wherein Zis D-Ser-Nle-D-Ser-D-Ser.
 29. The compound of claim 1, wherein Z isD-Ser-Lys-D-Ser-D-Ser.
 30. The compound of claim 1, wherein Z isD-Ser-Lys-D-Tyr-D-Tyr.
 31. The compound of claim 1, wherein Z isD-Ser-Lys-D-Tyr-D-Ser.
 32. The compound of claim 1, wherein Z isD-Ser-Ser-D-Lys-D-Ser.
 33. The compound of claim 1, wherein Z isD-Ser-Ser-D-Lys-Ser.
 34. The compound of claim 1, wherein Z isD-Ser-Nle-D-Tyr-D-Ser.
 35. The compound of claim 1, wherein Z isD-Ser-Pal-D-Tyr-D-Ser.
 36. The compound of claim 1, wherein Z isD-Ser-Thr-D-Tyr-D-Ser.
 37. The compound of claim 1, wherein Z isLys-D-Ser-D-Ser.
 38. The compound of claim 1, wherein Z isSer-D-Lys-D-Ser.
 39. The compound of claim 1, wherein Z isSer-D-Lys-Ser.
 40. The compound of claim 1, wherein Z isNle-D-Tyr-D-Ser.
 41. The compound of claim 1, wherein Z isLys-D-Tyr-D-Ser.
 42. The compound of claim 1, wherein Z isPal-D-Lys-D-Ser.
 43. The compound of claim 1, wherein Z isThr-D-Tyr-D-Ser.
 44. The compound of claim 1, wherein Z is D-Ser-D-Lys.45. The compound of claim 1, wherein Z is D-Ser-D-Tyr.
 46. The compoundof claim 1, wherein Z is D-Lys-D-Lys.
 47. The compound of claim 1,wherein Z is D-Lys-D-Tyr.
 48. The compound of claim 1, wherein Z isD-Tyr-D-Lys.
 49. The compound of claim 1, wherein Z has the formula:E-F, wherein: E is D-Lys, D-Tyr, D-Ser, D-4-OH-Pro, L-4-OH-Pro,3-iodo-D-Tyr, 3-5 diiodo-D-Tyr, 3-astatine-D-Tyr, 3-5 astatine-D-Tyr,3-bromo-D-Tyr, 3-5 dibromo-D-Tyr, D-Asn, L-Asn, D-Asp, L-Asp, D-Gln, orL-Gln; and F is D-Lys, D-Tyr, D-Ser, L-Ser, D-4-OH-Pro, L-4-OH-Pro,3-iodo-D Tyr, 3-5 diiodo-D-Tyr, 3-astatine-D-Tyr, 3-5 astatine-D-Tyr,3-bromo-D-Tyr, 3-5 dibromo-D-Tyr, D-Asn, L-Asn, D-Asp, L-Asp, D-Glu,L-Glu, D-Gln, or L-Gln.
 50. The compound of claim 1, wherein R comprises1 to 20 carbon atoms.
 51. The compound of claim 51, wherein R comprises1 to 8 carbon atoms.
 52. A method of treating a disease comprisingadministering to a subject suffering from said disease a therapeuticallyeffective amount of a compound having the formula:

wherein: X is a cytotoxic agent or therapeutic agent; n is an integerfrom 0 to 6, wherein (CH₂)_(n) is substituted or unsubstituted, astraight or branched chain, or is an alkyl, alkenyl, alkynyl, cyclic,heterocyclic, aromatic, or heteroaromatic group; R is N(R₁R₂), OR₁, orSR₁, wherein R₁ and R₂ are, independently, hydrogen or a lower alkylgroup; Y is a hydrophilic spacer sequence, or is omitted; Z is a linkingpeptide that, when bonded to Q at the N-terminus or at a compatibleside-chain amino group of Q, preserves at least 50% of the biologicalactivity of Q, Z having the formula: A-B-C-E-F, wherein: A is D-Lys,D-Tyr, D-Ser, or L-Ser, or is deleted; B is D-Lys or D-Tyr, or isdeleted; C is Lys, Ser, hSer, Thr, Nle, Abu, Nva, (2, 3, or 4)3-pyridyl-Ala (Pal), Orn, Dab, Dap, 4-NH₂-Phe, D-4-OH-Pro, orL-4-OH-Pro, or is deleted; E is D-Lys, D-Tyr, D-Ser, D-4-OH-Pro,L-4-OH-Pro, 3-iodo-D-Tyr, 3-5 diiodo-D-Tyr, 3-astatine-D-Tyr, 3-5astatine-D-Tyr, 3-bromo-D-Tyr, 3-5 dibromo-D-Tyr, D-Asn, L-Asn, D-Asp,L-Asp, D-Glu, L-Glu, D-Gln, or L-Gln; and F is D-Lys, D-Tyr, D-Ser,L-Ser, D-4-OH-Pro, L-4-OH-Pro, 3-iodo-D-Tyr, 3-5 diiodo-D-Tyr,3-astatine-D-Tyr, 3-5 astatine-D-Tyr, 3-bromo-D-Tyr, 3-5 dibromo-D-Tyr,D-Asn, L-Asn, D-Asp, L-Asp, D-Glu, L-Glu, D-Gln, or L-Gln; provided thatwhen A, B, C, and E are Tyr, Tyr, Lys, and Tyr, respectively, F is notLys; and when A, B, C, and E are Lys, Tyr, Lys, and Tyr, respectively, Eis not Tyr or Lys; and when A and B are deleted, and C and E are Lys andTyr, respectively, F is not Tyr or Lys, and Q is a targeting moiety, oris omitted.
 53. The method of claim 52, wherein X is a cytotoxic agent.54. The method of claim 53, wherein said cytotoxic agent iscamptothecin, homocamptothecin, colchicine, combretastatin, dolistatin,doxorubicin, methotrexate, podophyllotoxin, rhizoxin, rhizoxin D, ataxol, paclitaxol, CC1065, a maytansinoid, or derivatives or analogsthereof.
 55. The method of claim 52, wherein said hydrophilic spacersequence is a peptide that increases the hydrophilic biodistribution ofsaid compound.
 56. The method of claim 55, wherein said peptide has theformula U(V-V)_(n), wherein U is D-Pro, L-Pro, D-4-OH-Pro, L-4-OH-Pro,Sarcosine, Lys, Orn, Dab, Dap, 4-NH₂-Phe, or (NH₂—CH₂)_(m)—COOH), wherem=2-10, inclusive, or is deleted; each V is independently selected fromthe group consisting of: D-Ser, L-Ser, D-Thr, L-Thr, D-Gln, L-Gln,D-Asn, L-Asn, D-4-OH-Pro, or L-4 hydroxy-Pro; and n=1-50, inclusive. 57.The method of claim 56, wherein V is independently D-Ser or L-Ser. 58.The method of claim 56, wherein at least one V is a D-amino acid. 59.The method of claim 52, wherein said hydrophilic spacer sequence is ahydrophilic polymer.
 60. The method of claim 59, wherein saidhydrophilic polymer is polyethylene glycol, polyvinyl acetate, orpolyvinyl alcohol.
 61. The method of claim 52, wherein said targetingmoiety is a biologically-active peptide.
 62. The method of claim 61,wherein said biologically-active peptide is somatostatin, bombesin, aKiSS peptide, a urotensin II peptide, gonadotropin-releasing hormone(GnRH) I and II peptides, octreotide, depreotide, vapreotide, vasoactiveintestinal peptide (VIP), cholecystokinin (CCK), insulin-like growthfactor (IGF), RGD-containing peptides, melanocyte-stimulating hormone(MSH) peptide, neurotensin, calcitonin, a peptide comprising thecomplementarity determining region of an antitumor antibody,glutathione, a leukocyte-avid peptide comprising the amino acid sequenceYIGSR, the heparin-binding region of platelet factor-4 (PF-4), and alysine-rich sequence, atrial natriuretic peptide (ANP), a β-amyloidpeptide, a delta-opioid antagonist, annexin-V, endothelin, interleukin(IL)-1, IL-1ra, IL-2, IL-8, leukotriene B4 (LTB4), a chemotacticpeptide, a GP IIb/IIIa receptor antagonist, epidermal growth factor, ahuman neutrophil elastase inhibitor, plasmin inhibitor, an antimicrobialpeptide, apticide P280, apticide P274, a thrombospondin receptor,bitistatin, pituitary adenylyl cyclase type I receptor (PAC1), fibrinα-chain, or derivatives or analogs thereof.
 63. The method of claim 61,wherein said biologically-active peptide is an antibody or an antibodyfragment.
 64. The method of claim 62, wherein said leukocyte-avidpeptide is P483H.
 65. The method of claim 62, wherein said delta-opioidantagonist is ITIPP(psi).
 66. The method of claim 62, wherein saidchemotactic peptide is N-formyl-methionyl-leucyl-phenylalanine-lysine(fMLFK).
 67. The method of claim 62, wherein said GP IIb/IIIa receptorantagonist is DMP444.
 68. The method of claim 62, wherein said humanneutrophil elastase inhibitor is EPI-HNE-2 or EPI-HNE-4.
 69. The methodof claim 62, wherein said thrombospondin receptor is TP-1300.
 70. Themethod of claim 61, wherein said biologically-active peptide targetssaid compound to a cell or tissue in the body of a mammal.
 71. Themethod of claim 52, wherein said targeting moiety is a peptide derivedfrom a phage-display library, or a derivative or analog thereof, thattargets said compound to a cell or tissue in the body of a mammal. 72.The method of claim 70 or 71, wherein said cell or tissue comprises acancer cell, a white blood cell, cardiac tissue, brain tissue, or atubercle infected with tuberculosis.
 73. The method of claim 70 or 71,wherein said tissue is a tumor or a proliferative angiogenic bloodvessel.
 74. The method of claim 73, wherein said blood vessel is in theeye.
 75. The method of claim 50, wherein Q is somatostatin or an analogthereof, bombesin or an analog thereof, or an antibody.
 76. The methodof claim 52, wherein Q is a somatostatin peptide.
 77. The method ofclaim 52, wherein Q is a bombesin peptide.
 78. The method of claim 63,wherein said antibody is a monoclonal antibody.
 79. The method of claim52, wherein Z is D-Ser-Nle-D-Ser-D-Ser.
 80. The method of claim 52,wherein Z is D-Ser-Lys-D-Ser-D-Ser.
 81. The method of claim 52, whereinZ is D-Ser-Lys-D-Tyr-D-Tyr.
 82. The method of claim 52, wherein Z isD-Ser-Lys-D-Tyr-D-Ser.
 83. The method of claim 52, wherein Z isD-Ser-Ser-D-Lys-D-Ser.
 84. The method of claim 52, wherein Z isD-Ser-Ser-D-Lys-Ser.
 85. The method of claim 52, wherein Z isD-Ser-Nle-D-Tyr-D-Ser.
 86. The method of claim 52, wherein Z isD-Ser-Pal-D-Tyr-D-Ser.
 87. The method of claim 52, wherein Z isD-Ser-Thr-D-Tyr-D-Ser.
 88. The method of claim 52, wherein Z isLys-D-Ser-D-Ser.
 89. The method of claim 52, wherein Z isSer-D-Lys-D-Ser.
 90. The method of claim 52, wherein Z is Ser-D-Lys-Ser.91. The method of claim 52, wherein Z is Nle-D-Tyr-D-Ser.
 92. The methodof claim 52, wherein Z is Lys-D-Tyr-D-Ser.
 93. The method of claim 52,wherein Z is Pal-D-Lys-D-Ser.
 94. The method of claim 52, wherein Z isThr-D-Tyr-D-Ser.
 95. The method of claim 52, wherein Z is D-Ser-D-Lys.96. The method of claim 52, wherein Z is D-Ser-D-Tyr.
 97. The method ofclaim 52, wherein Z is D-Lys-D-Lys.
 98. The method of claim 52, whereinZ is D-Lys-D-Tyr.
 99. The method of claim 52, wherein Z is D-Tyr-D-Lys.100. The method of claim 52, wherein Z has the formula: E-F, wherein: Eis D-Lys, D-Tyr, D-Ser, D-4-OH-Pro, L-4-OH-Pro, 3-iodo-D-Tyr, 3-5diiodo-D-Tyr, 3-astatine-D-Tyr, 3-5 astatine-D-Tyr, 3-bromo-D-Tyr, 3-5dibromo-D-Tyr, D-Asn, L-Asn, D-Asp, L-Asp, D-Gln, or L-Gln; and F isD-Lys, D-Tyr, D-Ser, L-Ser, D-4-OH-Pro, L-4-OH-Pro, 3-iodo-D Tyr, 3-5diiodo-D-Tyr, 3-astatine-D-Tyr, 3-5 astatine-D-Tyr, 3-bromo-D-Tyr, 3-5dibromo-D-Tyr, D-Asn, L-Asn, D-Asp, L-Asp, D-Glu, L-Glu, D-Gln, orL-Gln.
 101. The method of claim 52, wherein said disease is inflammatorybowel disease, rheumatoid arthritis, acromegaly, tuberculosis, a tumorof the lung, breast, brain, eye, prostate or colon; a tumor ofneuroendocrine origin; or angiogenesis that causes inappropriateproliferation of blood vessels.
 102. The method of claim 101, whereinsaid tumor of neuroendocrine origin is carcinoid syndrome.
 103. Themethod of claim 101, wherein said blood vessels are in the eye.
 104. Themethod of claim 52, wherein comprises 1 to 20 carbon atoms.
 105. Themethod of claim 104, wherein R comprises 1 to 8 carbon atoms.
 106. Acompound represented by the following formula:

wherein: X is a cytotoxic agent or therapeutic agent; n is an integerfrom 0 to 6, wherein (CH₂)_(n) is substituted or unsubstituted, astraight or branched chain, or is an alkyl, alkenyl, alkynyl, cyclic,heterocyclic, aromatic, or heteroaromatic group; R is N(R₁R₂), OR₁, orSR₁, wherein R₁ and R₂ are, independently, hydrogen or a lower alkylgroup, and wherein R₃ is a NH(CH₂)mSH group and m=2 to 6, D or Lcysteine, a benzophenone, or an OH group.
 107. The compound of claim106, wherein the R₃ group can be used to attach a peptide, protein, orantibody to said compound.
 108. The compound of claim 106, wherein R₃ isNH(CH₂)mSH and m=2 to 6, and wherein said peptide, protein, or antibodyis attached to said compound by a thiol reaction.
 109. The compound ofclaim 106, wherein R₃ is a benzophenone and said peptide, protein, orantibody is attached to said compound by a photochemical reaction. 110.The compound of claim 109, wherein said benzophenone isp-benzoyl-phenylalanine.
 111. Use of a conjugate compound for themanufacture of a medicament for the treatment of a disease, saidcompound having the formula:

wherein: X is a cytotoxic agent or therapeutic agent; n is an integerfrom 0 to 6, wherein (CH₂)_(n) is substituted or unsubstituted, astraight or branched chain, or is an alkyl, alkenyl, alkynyl, cyclic,heterocyclic, aromatic, or heteroaromatic group; R is N(R₁R₂), OR₁, orSR₁, wherein R₁ and R₂ are, independently, hydrogen or a lower alkylgroup; Y is a hydrophilic spacer sequence, or is omitted; Z is a linkingpeptide that, when bonded to Q at the N-terminus or at a compatibleside-chain amino group of Q, preserves at least 50% of the biologicalactivity of Q, Z having the formula: A-B-C-E-F, wherein: A is D-Lys,D-Tyr, D-Ser, or L-Ser, or is deleted; B is D-Lys or D-Tyr, or isdeleted; C is Lys, Ser, hSer, Thr, Nle, Abu, Nva, (2, 3, or 4)3-pyridyl-Ala (Pal), Orn, Dab, Dap, 4-NH₂-Phe, D-4-OH-Pro, orL-4-OH-Pro, or is deleted; E is D-Lys, D-Tyr, D-Ser, D-4-OH-Pro,L-4-OH-Pro, 3-iodo-D-Tyr, 3-5 diiodo-D-Tyr, 3-astatine-D-Tyr, 3-5astatine-D-Tyr, 3-bromo-D-Tyr, 3-5 dibromo-D-Tyr, D-Asn, L-Asn, D-Asp,L-Asp, D-Glu, L-Glu, D-Gln, or L-Gln; and F is D-Lys, D-Tyr, D-Ser,L-Ser, D-4-OH-Pro, L-4-OH-Pro, 3-iodo-D-Tyr, 3-5 diiodo-D-Tyr,3-astatine-D-Tyr, 3-5 astatine-D-Tyr, 3-bromo-D-Tyr, 3-5 dibromo-D-Tyr,D-Asn, L-Asn, D-Asp, L-Asp, D-Glu, L-Glu, D-Gln, or L-Gln; provided thatwhen A, B, C, and E are Tyr, Tyr, Lys, and Tyr, respectively, F is notLys; and when A, B, C, and E are Lys, Tyr, Lys, and Tyr, respectively, Eis not Tyr or Lys; and when A and B are deleted, and C and E are Lys andTyr, respectively, F is not Tyr or Lys, and Q is a targeting moiety, oris omitted.
 112. The use of claim 111, wherein said disease isinflammatory bowel disease, rheumatoid arthritis, acromegaly,tuberculosis, a tumor of the lung, breast, brain, eye, prostate orcolon; a tumor of neuroendocrine origin; or angiogenesis that causesinappropriate proliferation of blood vessels.
 113. The use of claim 111,wherein said tumor of neuroendocrine origin is carcinoid syndrome. 114.The use of claim 111, wherein said blood vessels are in the eye.